Targeted therapeutics

ABSTRACT

The present invention provides pharmacological compounds including an effector moiety conjugated to a binding moiety that directs the effector moiety to a biological target of interest. Likewise, the present invention provides compositions, kits, and methods (e.g., therapeutic, diagnostic, and imaging) including the compounds. The compounds can be described as a protein interacting binding moiety-drug conjugate (SDC-TRAP) compounds, which include a protein interacting binding moiety and an effector moiety. For example, in certain embodiments directed to treating cancer, the SDC-TRAP can include an Hsp90 inhibitor conjugated to a cytotoxic agent as the effector moiety.

RELATED APPLICATIONS

This application is a continuation of International Application No.PCT/US2014/062689, filed on Oct. 28, 2014, which claims priority to U.S.Provisional Application No. 61/896,416, filed on Oct. 28, 2013, theentire contents of which are incorporated by reference herein.

FIELD OF THE INVENTION

The present invention relates to pharmacological compounds including aneffector moiety conjugated to a binding moiety that directs the effectormoiety to a biological target of interest. The compounds have broadpharmacological applications, including therapeutics, diagnostics, andimaging. For example, the compounds can specifically direct therapeuticeffector moieties to target cells or tissue of interest, for targetedchemotherapeutic treatment of conditions such as cancer.

BACKGROUND OF THE INVENTION

Although tremendous advances have been made in chemotherapy, currentlyavailable therapeutics and therapies remain unsatisfactory and theprognosis for the majority of patients diagnosed withchemotherapeutically treated diseases (e.g., cancer) remains poor.Often, the applicability and/or effectiveness of chemotherapy, as wellas other therapies and diagnostics employing potentially toxic moieties,is limited by undesired side effects.

Many disease and disorders are characterized by the presence of highlevels of certain proteins in specific types of cells. In some cases,the presence of these high levels of protein is caused byoverexpression. Historically, some of these proteins have been usefultargets for therapeutic molecules or used as biomarkers for thedetection of disease. One class of overexpressed intracellular proteinthat has been recognized as a useful therapeutic target is known as theheat shock proteins.

Heat shock proteins (HSPs) are a class of proteins that are up-regulatedin response to elevated temperature and other environmental stresses,such as ultraviolet light, nutrient deprivation, and oxygen deprivation.HSPs have many known functions, including acting as chaperones to othercellular proteins (called client proteins) to facilitate their properfolding and repair, and to aid in the refolding of misfolded clientproteins. There are several known families of HSPs, each having its ownset of client proteins. Hsp90 is one of the most abundant HSP families,accounting for about 1-2% of proteins in a cell that is not under stressand increasing to about 4-6% in a cell under stress.

Inhibition of Hsp90 results in degradation of its client proteins viathe ubiquitin proteasome pathway. Unlike other chaperone proteins, theclient proteins of Hsp90 are mostly protein kinases or transcriptionfactors involved in signal transduction, and a number of its clientproteins have been shown to be involved in the progression of cancer.Hsp90 has been shown by mutational analysis to be necessary for thesurvival of normal eukaryotic cells. However, Hsp90 is overexpressed inmany tumor types, indicating that it may play a significant role in thesurvival of cancer cells and that cancer cells may be more sensitive toinhibition of Hsp90 than normal cells. For example, cancer cellstypically have a large number of mutated and overexpressed oncoproteinsthat are dependent on Hsp90 for folding. In addition, because theenvironment of a tumor is typically hostile due to hypoxia, nutrientdeprivation, acidosis, etc., tumor cells may be especially dependent onHsp90 for survival. Moreover, inhibition of Hsp90 causes simultaneousinhibition of a number of oncoproteins, as well as hormone receptors andtranscription factors, making it an attractive target for an anti-canceragent. In view of the above, Hsp90 has been an attractive target of drugdevelopment, including such Hsp90 inhibitor (Hsp90i) compounds asganetespib, AUY-922, and IPI-504. At the same time, the advancement ofcertain of these compounds which showed early promise, e.g.,geldanamycin, has been slowed by those compounds' toxicity profile.Hsp90i compounds developed to date are believed to show great promise ascancer drugs, but other ways the ubiquity of Hsp90 in cancer cells mightbe leveraged have heretofore remained unexplored until now. Accordingly,the need exists for therapeutic molecules that selectively targetproteins, such as Hsp90, that are overexpressed in cells associated withparticular diseases or disorders.

SUMMARY OF THE INVENTION

The present invention provides pharmacological molecules (“SDC-TRAPs”).I none aspect, the invention features a binding moiety-drug conjugate(SDC-TRAP) comprising a binding moiety and an effector moiety, forexample and including an effector moiety conjugated to a binding moiety,which directs the effector moiety into a target cell of interest in amanner that traps the molecule in the target cell. In a specificembodiment, the effector moiety is conjugated via a cleavable bond orlinker to the binding moiety, such that the cleavable bond or linker ispreferentially cleaved after the SDC-TRAP enters the target cell. Theinventors of the instant application have discovered that the SDC-TRAPmolecules of the invention can be used to selectively deliver aneffector moiety to a specific type of cell in order to increase theintracellular level of the effector moiety in the target cell ascompared to other cells. In one embodiment, the binding moiety interactswith a protein that is overexpressed in cancerous cells compared tonormal cells. The inventors have demonstrated that certain SDC-TRAPmolecules of the invention enter target cells by passive diffusion andare selectively retained in the target cells. Specifically, theinventors have shown that certain SDC-TRAP molecules of the inventionare selectively retained only in cells that overexpress or otherwisehave a high intracellular level of the protein to which the bindingmoiety binds. There are numerous advantages to these SDC-TRAP moleculesand to methods of using these molecules that are described herein.

Specifically, the invention provides SDC-TRAP molecules that aretargeted to cells of interest and trapped intracellularly for asufficient period of time such that the effector moiety has the desiredbiological effect. In one embodiment, these SDC-TRAPs allow for thetargeting of an effector moiety to a particular type of cell based onthe overexpression of an intracellular protein that is characteristic ofa particular disease or disorder. Accordingly, the present inventionprovides compositions, kits, and methods (e.g., therapeutic, diagnostic,and imaging) including the compounds.

In a specific embodiment, the application exemplifies the use of Hsp90interacting moieties, e.g., inhibitors, as the binding moiety in theSDC-TRAPs. However, the invention is intended to include other bindingmoieties, including those that are contemplated, listed and exemplifiedherein. Accordingly, in certain embodiments directed to treating canceror inflammation, the SDC-TRAP includes an Hsp90 inhibitor moietyconjugated to an effector moiety. In certain embodiments, the effectormoiety is a cytotoxic effector moiety.

In another embodiment, the SDC-TRAP includes an effector moiety that iseffective while still linked to the binding moiety. In such embodiment,cleavage of the bond or linker in the target cell is not a necessaryfeature of the invention. In other cases, such as cytotoxic effectormoieties, the effector moiety should only be effective after the linkeror bond is cleaved and the effector moiety is released from the SDC-TRAPmolecule inside the target cell. In either case, SDC-TRAPs that do notenter into the target cell should be rapidly cleared (e.g., from theplasma or other non-target cells or tissues).

In another embodiment, the binding moiety of the SDC-TRAP binds aprotein within the target cell, which may itself produce a desiredbiological effect (e.g., such as inhibiting Hsp90 within the targetcell). In one embodiment, the binding moiety can contribute to theoverall efficacy of the SDC-TRAP by not only binding an intracellularprotein present in the target cell but by also conveying a particulardesired biological effect. For example, if the binding moiety is anHsp90 inhibitor and the target cell is a cancer cell, than the overallactivity of the SDC-TRAP may not only result from the effector moiety,but also from the biological activity of the Hsp90 inhibitor.

Alternatively, interaction of the binding moiety with its protein targetmay not impart a biological effect, but rather only serve to attract andretain the SDC-TRAP within the target cell. In this embodiment, thebinding moiety may reversibly bind to the intracellular target proteinand create an intracellular equilibrium between free and bound SDC-TRAPmolecules. This equilibrium may allow for cleavage of the SDC-TRAP andmore effective delivery of the effector moiety, e.g., release of theeffector moiety from the binding moiety by, for example, enzymaticcleavage, hydrolysis or degradation. In some cases, the effector moietymay be inactive until such release occurs.

In various aspects and embodiments, the present invention providesnumerous advantages. For example, the SDC-TRAP can provide for targetedtherapy, maximizing efficacy and/or minimizing undesired side effects.The SDC-TRAP can provide for targeted use of an effector moiety thatwould otherwise be unsuitable for administration alone due to toxicityand/or undesired systemic effects. The SDC-TRAP can facilitate targetingsuch effector moieties to intracellular targets—that is, due to its sizeand chemical properties, the SDC-TRAP can passively diffuse (or in somecases be actively transported) into a cell having an intracellulartarget of interest. Alternatively, the SDC-TRAP can deliver in aselective manner a cytotoxic molecule to destroy a target cell, such asa cancer or inflammatory cell.

In various aspects and embodiments, the SDC-TRAP can exhibit decreasedand/or minimized toxicity concurrently with increased efficacy (e.g., ascompared to that of the effector moiety when used alone). Decreasingand/or minimizing toxicity can encompass reducing toxicity to apredetermined level (e.g., a regulatory guideline or suggested level,for example promulgated by the US Food and Drug Administration “FDA”).Increasing efficacy can encompass increasing efficacy to a predeterminedlevel (e.g., a regulatory guideline or suggested level, for examplepromulgated by the US FDA). Similarly, decreasing and/or minimizingtoxicity concurrently with increasing efficacy can encompass achieving apredetermined therapeutic ratio (e.g., a regulatory guideline orsuggested value, for example promulgated by the US FDA).

Decreasing and/or minimizing toxicity can encompass, for example,reducing toxicity by 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, 95%, or more. Increasing efficacy can encompass, forexample, increasing efficacy by 5, 10, 15, 20, 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 250, 300,400, 500%, or more. Decreasing and/or minimizing toxicity concurrentlywith increasing efficacy can encompass, for example: essentially thesame efficacy with decreased toxicity; essentially the same toxicitywith increased efficacy; or decreased toxicity and increased efficacy.Similarly, decreasing and/or minimizing toxicity concurrently withincreasing efficacy can encompass, for example, scenarios such as:increased efficacy enabling a lower dose (e.g., lower dose of effectormoiety with a correspondingly lower net toxicity) and decreased toxicityenabling a higher dose (e.g., higher dose of effector moiety without acorrespondingly higher net toxicity).

Additional advantages are discussed in detail below.

These and other advantages of the present invention are of particularinterest, for example, in chemotherapy where despite tremendous recentadvances, currently available therapeutics and therapies remainsunsatisfactory and the prognosis for the majority of patients diagnosedwith diseases such as cancer remains poor. However, while many of theillustrative embodiments and examples are presented in the context ofcancer, a person of ordinary skill in the art would understand that thepresent invention has applications across therapeutic, diagnostic, andimaging applications that require, or would benefit from, targeting ofan effector moiety.

In various aspects, the invention provides an SDC-TRAP comprising abinding moiety and an effector moiety.

In various aspects, the invention provides an SDC-TRAP comprising abinding moiety and an effector moiety, wherein the SDC-TRAP is able toenter a cell by active transport.

In various aspects, the invention provides an SDC-TRAP comprising abinding moiety and an effector moiety, wherein the SDC-TRAP has amolecular weight of less than about 1600 Daltons.

In various aspects, the invention provides an SDC-TRAP comprising abinding moiety and an effector moiety, wherein the binding moiety has amolecular weight of less than about 800 Daltons.

In various aspects, the invention provides an SDC-TRAP comprising abinding moiety and an effector moiety, wherein the effector moiety has amolecular weight of less than 800 Daltons.

In various aspects, the invention provides an SDC-TRAP comprising abinding moiety and an effector moiety, wherein the binding moiety andthe effector moiety are approximately equal in size.

In various aspects, the invention provides an SDC-TRAP comprising anHsp90 binding moiety and an effector moiety, wherein the Hsp90 bindingmoiety interacts with the N-terminal domain of Hsp90.

In various aspects, the invention provides an SDC-TRAP comprising anHsp90 binding moiety and an effector moiety, wherein the Hsp90 bindingmoiety interacts with the C-terminal domain of Hsp90.

In various aspects, the invention provides an SDC-TRAP comprising anHsp90 binding moiety and an effector moiety, wherein the Hsp90 bindingmoiety interacts with the middle domain of Hsp90.

In various aspects, the invention provides an SDC-TRAP comprising abinding moiety and an effector moiety, wherein the binding moietyinteracts with a predetermined domain of a multidomain target proteinmolecule.

In various aspects, the invention provides an SDC-TRAP comprising abinding moiety (e.g., an Hsp90 binding moiety) and an effector moiety,wherein the binding moiety (e.g., Hsp90 binding moiety) has a K_(d) of100 nM or higher (e.g., for a predetermined target molecule, forexample, Hsp90).

In various aspects, the invention provides an SDC-TRAP comprising abinding moiety (e.g., Hsp90 binding moiety) and an effector moiety,wherein when administered to a subject, the SDC-TRAP is present at aratio of 2:1 in target (e.g., tumor) cells compared to plasma. Inanother embodiment, the invention provides an SDC-TRAP comprising abinding moiety (e.g., Hsp90 binding moiety) and an effector moiety,wherein when administered to a subject the SDC-TRAP present at a ratioof 2:1 in target (e.g., tumor) cells compared to normal cells.

In various aspects, the invention provides an SDC-TRAP comprising abinding moiety (e.g., Hsp90 binding moiety) and an effector moiety,wherein the SDC-TRAP is present in target (e.g., cancer) cells for atleast 24 hours.

In various aspects, the invention provides an SDC-TRAP comprising abinding moiety (e.g., Hsp90 binding moiety) and an effector moiety,wherein the effector moiety is released for a period of at least 6 hours(e.g., within a target cell and/or tissue).

In various aspects, the invention provides an SDC-TRAP comprising abinding moiety (e.g., Hsp90 binding moiety) and an effector moiety,wherein the effector moiety is selectively released inside a target(e.g., cancer) cell.

In various aspects, the invention provides an SDC-TRAP comprising abinding moiety (e.g., Hsp90 binding moiety) and an effector moiety,wherein the SDC-TRAP allows for the use of an effector moiety that istoxic or otherwise unfit for administration to a subject.

In various aspects, the invention provides an SDC-TRAP comprising abinding moiety (e.g., Hsp90 binding moiety) and an effector moiety,wherein the Hsp90 is an inhibitor (e.g., Hsp90 inhibitor) that isineffective as a therapeutic agent when administered alone.

In various aspects, the invention provides an SDC-TRAP comprising anHsp90 binding moiety and an effector moiety.

In various aspects, the invention provides pharmaceutical compositionscomprising a therapeutically effective amount of at least one SDC-TRAP,and at least one pharmaceutical excipient.

In various aspects, the invention provides methods for treating asubject in need thereof comprising administering a therapeuticallyeffective amount of at least one SDC-TRAP to the subject, therebytreating the subject.

In various aspects, the invention provides methods for imaging,diagnosing, and/or selecting a subject comprising administering aneffective amount of at least one SDC-TRAP to the subject, therebyimaging, diagnosing, and/or selecting the subject.

In various aspects, the invention provides kits for treating a subjectin need thereof comprising at least one SDC-TRAP and instruction foradministering a therapeutically effective amount of the at least oneSDC-TRAP to the subject, thereby treating the subject.

In various aspects, the invention provides kits for imaging, diagnosing,and/or selecting a subject comprising at least one SDC-TRAP andinstruction for administering an effective amount of at least oneSDC-TRAP to the subject, thereby imaging, diagnosing, and/or selectingthe subject.

In various embodiments, the invention can include any one or more of theaspects disclosed herein having any one or more of the featuresdisclosed herein.

In various embodiments, the binding moiety interacts with a protein thatis overexpressed in cancerous cells compared to normal cells.

In various embodiments, the protein is a chaperonin protein. Thechaperonin can be, for example, Hsp90.

In various embodiments, the chaperonin is an Hsp90 binding moiety.

In various embodiments, the binding moiety is an Hsp90 ligand or aprodrug thereof. The Hsp90 ligand can be, for example, an Hsp90inhibitor. An Hsp90 inhibitor can be selected from the group consistingof geldanamycins, macbecins, tripterins, tanespimycins, and radicicols.

In various embodiments, the binding moiety can be an Hsp90-targetingmoiety, for example a triazole/resorcinol-based compound that bindsHsp90, or a resorcinol amide-based compound that binds Hsp90, e.g.,ganetespib, AUY-922, or AT-13387.

In various embodiments, the binding moiety can be an Hsp90-bindingcompound of formula (I):

wherein

R¹ may be alkyl, aryl, halide, carboxamide or sulfonamide; R² may bealkyl, cycloalkyl, aryl or heteroaryl, wherein when R² is a six-memberedaryl or heteroaryl, R² is substituted at the 3- and 4-positions relativeto the connection point on the triazole ring, through which a linker Lis attached; and R³ may be SH, OH, —CONHR⁴, aryl or heteroaryl, whereinwhen R³ is a six-membered aryl or heteroaryl, R³ is substituted at the 3or 4 position.

In various embodiments, the binding moiety can be an Hsp90-bindingcompound of formula (II):

wherein

R¹ may be alkyl, aryl, halo, carboxamido, sulfonamido; and R² may beoptionally substituted alkyl, cycloalkyl, aryl or heteroaryl. Examplesof such compounds include5-(2,4-dihydroxy-5-isopropylphenyl)-N-(2-morpholinoethyl)-4-(4-(morpholinomethyl)phenyl)-4H-1,2,4-triazole-3-carboxamideand5-(2,4-dihydroxy-5-isopropylphenyl)-4-(4-(4-methylpiperazin-1-yl)phenyl)-N-(2,2,2-trifluoroethyl)-4H-1,2,4-triazole-3-carboxamide.

In various embodiments, the binding moiety can be an Hsp90-bindingcompound of formula (III):

wherein

X, Y, and Z may independently be CH, N, O or S (with appropriatesubstitutions and satisfying the valency of the corresponding atoms andaromaticity of the ring); R¹ may be alkyl, aryl, halide, carboxamido orsulfonamido; R² may be substituted alkyl, cycloalkyl, aryl orheteroaryl, where a linker L is connected directly or to the extendedsubstitutions on these rings; R³ may be SH, OH, NR⁴R⁵ AND —CONHR⁶, towhich an effector moiety may be connected; R⁴ and R⁵ may independentlybe H, alkyl, aryl, or heteroaryl; and R⁶ may be alkyl, aryl, orheteroaryl, having a minimum of one functional group to which aneffector moiety may be connected.

As used herein, the term “alkyl” means a saturated straight chain orbranched non-cyclic hydrocarbon having from 1 to 10 carbon atoms.Representative saturated straight chain alkyls include methyl, ethyl,n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl andn-decyl; while saturated branched alkyls include isopropyl, sec-butyl,isobutyl, tert-butyl, isopentyl, 2-methylbutyl, 3-methylbutyl,2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2-methylhexyl,3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2,3-dimethylbutyl,2,3-dimethylpentyl, 2,4-dimethylpentyl, 2,3-dimethylhexyl,2,4-dimethylhexyl, 2,5-dimethylhexyl, 2,2-dimethylpentyl,2,2-dimethylhexyl, 3,3-dimtheylpentyl, 3,3-dimethylhexyl,4,4-dimethylhexyl, 2-ethylpentyl, 3-ethylpentyl, 2-ethylhexyl,3-ethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl,2-methyl-3-ethylpentyl, 2-methyl-4-ethylpentyl, 2-methyl-2-ethylhexyl,2-methyl-3-ethylhexyl, 2-methyl-4-ethylhexyl, 2,2-diethylpentyl,3,3-diethylhexyl, 2,2-diethylhexyl, 3,3-diethylhexyl and the like. Theterm “(C₁-C₆)alkyl” means a saturated straight chain or branchednon-cyclic hydrocarbon having from 1 to 6 carbon atoms. Representative(C₁-C₆)alkyl groups are those shown above having from 1 to 6 carbonatoms. Alkyl groups included in compounds of this invention may beoptionally substituted with one or more substituents.

As used herein, the term “alkenyl” means a saturated straight chain orbranched non-cyclic hydrocarbon having from 2 to 10 carbon atoms andhaving at least one carbon-carbon double bond. Representative straightchain and branched (C₂-C₁₀)alkenyls include vinyl, allyl, 1-butenyl,2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl,2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, 1-hexenyl, 2-hexenyl,3-hexenyl, 1-heptenyl, 2-heptenyl, 3-heptenyl, 1-octenyl, 2-octenyl,3-octenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 1-decenyl, 2-decenyl,3-decenyl and the like. Alkenyl groups may be optionally substitutedwith one or more substituents.

As used herein, the term “alkynyl” means a saturated straight chain orbranched non-cyclic hydrocarbon having from 2 to 10 carbon atoms andhaving at least one carbon-carbon triple bond. Representative straightchain and branched alkynyls include acetylenyl, propynyl, 1-butynyl,2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1-butynyl, 4-pentynyl,1-hexynyl, 2-hexynyl, 5-hexynyl, 1-heptynyl, 2-heptynyl, 6-heptynyl,1-octynyl, 2-octynyl, 7-octynyl, 1-nonynyl, 2-nonynyl, 8-nonynyl,1-decynyl, 2-decynyl, 9-decynyl, and the like. Alkynyl groups may beoptionally substituted with one or more substituents.

As used herein, the term “cycloalkyl” means a saturated, mono- orpolycyclic alkyl radical having from 3 to 20 carbon atoms.Representative cycloalkyls include cyclopropyl, 1-methylcyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl,cyclononyl, -cyclodecyl, octahydro-pentalenyl, and the like. Cycloalkylgroups may be optionally substituted with one or more substituents.

As used herein, the term “cycloalkenyl” means a mono- or poly-cyclicnon-aromatic alkyl radical having at least one carbon-carbon double bondin the cyclic system and from 3 to 20 carbon atoms. Representativecycloalkenyls include cyclopentenyl, cyclopentadienyl, cyclohexenyl,cyclohexadienyl, cycloheptenyl, cycloheptadienyl, cycloheptatrienyl,cyclooctenyl, cyclooctadienyl, cyclooctatrienyl, cyclooctatetraenyl,cyclononenyl, cyclononadienyl, cyclodecenyl, cyclodecadienyl,1,2,3,4,5,8-hexahydronaphthalenyl and the like. Cycloalkenyl groups maybe optionally substituted with one or more substituents.

As used herein, the term “haloalkyl” means and alkyl group in which oneor more (including all) the hydrogen radicals are replaced by a halogroup, wherein each halo group is independently selected from —F, —Cl,—Br, and —I. The term “halomethyl” means a methyl in which one to threehydrogen radical(s) have been replaced by a halo group. Representativehaloalkyl groups include trifluoromethyl, bromomethyl,1,2-dichloroethyl, 4-iodobutyl, 2-fluoropentyl, and the like.

As used herein, an “alkoxy” is an alkyl group which is attached toanother moiety via an oxygen linker.

As used herein, an “haloalkoxy” is an haloalkyl group which is attachedto another moiety via an oxygen linker.

As used herein, the term an “aromatic ring” or “aryl” means ahydrocarbon monocyclic or polycyclic radical in which at least one ringis aromatic. Examples of suitable aryl groups include, but are notlimited to, phenyl, tolyl, anthracenyl, fluorenyl, indenyl, azulenyl,and naphthyl, as well as benzo-fused carbocyclic moieties such as5,6,7,8-tetrahydronaphthyl. Aryl groups may be optionally substitutedwith one or more substituents. In one embodiment, the aryl group is amonocyclic ring, wherein the ring comprises 6 carbon atoms, referred toherein as “(C₆)aryl.”

As used herein, the term “aralkyl” means an aryl group that is attachedto another group by a (C₁-C₆)alkylene group. Representative aralkylgroups include benzyl, 2-phenyl-ethyl, naphth-3-yl-methyl and the like.Aralkyl groups may be optionally substituted with one or moresubstituents.

As used herein, the term “alkylene” refers to an alkyl group that hastwo points of attachment. The term “(C₁-C₆)alkylene” refers to analkylene group that has from one to six carbon atoms. Straight chain(C₁-C₆)alkylene groups are preferred. Non-limiting examples of alkylenegroups include methylene (—CH₂—), ethylene (—CH₂CH₂—), n-propylene(—CH₂CH₂CH₂—), isopropylene (—CH₂CH(CH₃)—), and the like. Alkylenegroups may be optionally substituted with one or more substituents.

As used herein, the term “heterocyclyl” means a monocyclic (typicallyhaving 3- to 10-members) or a polycyclic (typically having 7- to20-members) heterocyclic ring system which is either a saturated ring ora unsaturated non-aromatic ring. A 3- to 10-membered heterocycle cancontain up to 5 heteroatoms; and a 7- to 20-membered heterocycle cancontain up to 7 heteroatoms. Typically, a heterocycle has at least oncarbon atom ring member. Each heteroatom is independently selected fromnitrogen, which can be oxidized (e.g., N(O)) or quaternized; oxygen; andsulfur, including sulfoxide and sulfone. The heterocycle may be attachedvia any heteroatom or carbon atom. Representative heterocycles includemorpholinyl, thiomorpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl,piperazinyl, hydantoinyl, valerolactamyl, oxiranyl, oxetanyl,tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyrindinyl,tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, andthe like. A heteroatom may be substituted with a protecting group knownto those of ordinary skill in the art, for example, the hydrogen on anitrogen may be substituted with a tert-butoxycarbonyl group.Furthermore, the heterocyclyl may be optionally substituted with one ormore substituents. Only stable isomers of such substituted heterocyclicgroups are contemplated in this definition.

As used herein, the term “heteroaromatic”, “heteroaryl” or like termsmeans a monocyclic or polycyclic heteroaromatic ring comprising carbonatom ring members and one or more heteroatom ring members. Eachheteroatom is independently selected from nitrogen, which can beoxidized (e.g., N(O)) or quaternized; oxygen; and sulfur, includingsulfoxide and sulfone. Representative heteroaryl groups include pyridyl,1-oxo-pyridyl, furanyl, benzo[1,3]dioxolyl, benzo[1,4]dioxinyl, thienyl,pyrrolyl, oxazolyl, imidazolyl, thiazolyl, a isoxazolyl, quinolinyl,pyrazolyl, isothiazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, atriazinyl, triazolyl, thiadiazolyl, isoquinolinyl, indazolyl,benzoxazolyl, benzofuryl, indolizinyl, imidazopyridyl, tetrazolyl,benzimidazolyl, benzothiazolyl, benzothiadiazolyl, benzoxadiazolyl,indolyl, tetrahydroindolyl, azaindolyl, imidazopyridyl, quinazolinyl,purinyl, pyrrolo[2,3]pyrimidinyl, pyrazolo[3,4]pyrimidinyl,imidazo[1,2-a]pyridyl, and benzothienyl. In one embodiment, theheteroaromatic ring is selected from 5-8 membered monocyclic heteroarylrings. The point of attachment of a heteroaromatic or heteroaryl ring toanother group may be at either a carbon atom or a heteroatom of theheteroaromatic or heteroaryl rings. Heteroaryl groups may be optionallysubstituted with one or more substituents.

As used herein, the term “(C₅)heteroaryl” means an aromatic heterocyclicring of 5 members, wherein at least one carbon atom of the ring isreplaced with a heteroatom such as, for example, oxygen, sulfur ornitrogen. Representative (C₅)heteroaryls include furanyl, thienyl,pyrrolyl, oxazolyl, imidazolyl, thiazolyl, isoxazolyl, pyrazolyl,isothiazolyl, pyrazinyl, triazolyl, thiadiazolyl, and the like.

As used herein, the term “(C₆)heteroaryl” means an aromatic heterocyclicring of 6 members, wherein at least one carbon atom of the ring isreplaced with a heteroatom such as, for example, oxygen, nitrogen orsulfur. Representative (C₆)heteroaryls include pyridyl, pyridazinyl,pyrazinyl, triazinyl, tetrazinyl and the like.

As used herein, the term “heteroaralkyl” means a heteroaryl group thatis attached to another group by a (C₁-C₆)alkylene. Representativeheteroaralkyls include 2-(pyridin-4-yl)-propyl, 2-(thien-3-yl)-ethyl,imidazol-4-yl-methyl and the like. Heteroaralkyl groups may beoptionally substituted with one or more substituents.

As used herein, the term “halogen” or “halo” means —F, —Cl, —Br or —I.

Suitable substituents for an alkyl, alkylene, alkenyl, alkynyl,cycloalkyl, cycloalkenyl, heterocyclyl, aryl, aralkyl, heteroaryl, andheteroaralkyl groups include any substituent which will form a stablecompound of the invention. Examples of substituents for an alkyl,alkylene, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocyclyl,aryl, aralkyl, heteroaryl, and heteroarylalkyl include an optionallysubstituted alkyl, an optionally substituted alkenyl, an optionallysubstituted alkynyl, an optionally substituted cycloalkyl, an optionallysubstituted cycloalkenyl, an optionally substituted heterocyclyl, anoptionally substituted aryl, an optionally substituted heteroaryl, anoptionally substituted aralkyl, an optionally substituted heteraralkyl,or a haloalkyl.

In addition, alkyl, cycloalkyl, alkylene, a heterocyclyl, and anysaturated portion of a alkenyl, cycloalkenyl, alkynyl, aralkyl, andheteroaralkyl groups, may also be substituted with ═O, or ═S.

When a heterocyclyl, heteroaryl, or heteroaralkyl group contains anitrogen atom, it may be substituted or unsubstituted. When a nitrogenatom in the aromatic ring of a heteroaryl group has a substituent thenitrogen may be a quaternary nitrogen.

As used herein, the term “lower” refers to a group having up to fouratoms. For example, a “lower alkyl” refers to an alkyl radical havingfrom 1 to 4 carbon atoms, “lower alkoxy” refers to “—O—(C₁-C₄)alkyl anda “lower alkenyl” or “lower alkynyl” refers to an alkenyl or alkynylradical having from 2 to 4 carbon atoms, respectively.

Unless indicated otherwise, the compounds of the invention containingreactive functional groups (such as (without limitation) carboxy,hydroxy, thiol, and amino moieties) also include protected derivativesthereof. “Protected derivatives” are those compounds in which a reactivesite or sites are blocked with one or more protecting groups. Examplesof suitable protecting groups for hydroxyl groups include benzyl,methoxymethyl, allyl, trimethylsilyl, tert-butyldimethylsilyl, acetate,and the like. Examples of suitable amine protecting groups includebenzyloxycarbonyl, tert-butoxycarbonyl, tert-butyl, benzyl andfluorenylmethyloxy-carbonyl (Fmoc). Examples of suitable thiolprotecting groups include benzyl, tert-butyl, acetyl, methoxymethyl andthe like. Other suitable protecting groups are well known to those ofordinary skill in the art and include those found in T. W. Greene,Protecting Groups in Organic Synthesis, John Wiley & Sons, Inc. 1981.

Exemplary Hsp90 inhibitors include those disclosed in U.S. Pat. Nos.8,362,055 and 7,825,148. Examples of such compounds include AUY-922:

In various embodiments, the binding moiety can be an Hsp90-bindingcompound of formula (IV):

wherein

R¹ may be alkyl, aryl, halo, carboxamido or sulfonamido; R² and R³ areindependently C₁-C₅ hydrocarbyl groups optionally substituted with oneor more of hydroxy, halogen, C₁-C₂ alkoxy, amino, mono- and di-C₁-C₂alkylamino; 5- to 12-membered aryl or heteroaryl groups; or, R² and R³,taken together with the nitrogen atom to which they are attached, form a4- to 8-membered monocyclic heterocyclic group, of which up to 5 ringmembers are selected from O, N and S. Examples of such compounds includeAT-13387:

In various embodiments, the binding moiety includes an Hsp90-targetingmoiety, for example one or more geldanamycins, e.g., IPI-493

macbecins, tripterins, tanespimycins, e.g., 17-AAG

KF-55823

radicicols, KF-58333

KF-58332

17-DMAG

IPI 504

BIIB-021

BBB-028, PU-H64

PU-H71

PU-DZ8

PU-HZ151

SNX-2112

SNX-2321

SNX-5422

SNX-7081

SNX-8891, SNX-0723

SAR-567530, ABI-287, ABI-328, AT-13387

NSC-113497

PF-3823863

PF-4470296

EC-102, EC-154, ARQ-250-RP, BC-274

VER-50589

KW-2478

BHI-001, AUY-922

EMD-614684

EMD-683671, XL-888, VER-51047

KOS-2484, KOS-2539, CUDC-305

MPC-3100

CH-5164840

PU-DZ13

PU-HZ151

PU-DZ13

VER-82576

VER-82160

VER-82576

VER-82160

NXD-30001

NVP-HSP990

SST-0201CL1

SST-0115AA1

SST-0221AA1

SST-0223AA1

novobiocin (a C-terminal Hsp90i.)

In various embodiments, the cytotoxic moiety is selected from the groupconsisting of LY2801653

LY2835219

LY2606368

LY2874455

LY2090314

LY2940680

LY278544

LY2228820

LY2157299

LY2603618, LY353381

and trimetrexate

In various embodiments, the cytotoxic moiety is not suitable foradministration alone. The cytotoxic moiety can be unsuitable foradministration alone due to toxicity. The cytotoxic moiety can beunsuitable for administration alone due to undesired targeting or a lackof targeting.

In various embodiments, the binding moiety and the effector moiety arecovalently attached. The binding moiety and the effector moiety can becovalently attached, for example by a linker. The linker can comprise acleavable linker. The cleavable linker can comprise an enzymaticallycleavable linker. The linker can be selected from the group consistingof disulfide, carbamate, amide, ester, and ether linkers.

In various embodiments, the SDC-TRAP has a molecular weight of less thanabout 1600 Dalton. For example, the SDC-TRAP molecular weight can beless than about 1600, 1550, 1500, 1450, 1400, 1350, 1300, 1250, 1200,1150, 1100, 1050, 1000, 950, 900, 850, 800, 750, 700, 650, 600, 550,500, 450, 400, 350, 300, 250, or 200 Dalton.

In various embodiments, the binding moiety has a molecular weight ofless than about 800 Dalton. For example, the binding moiety molecularweight can be less than about 800, 750, 700, 650, 600, 550, 500, 450,400, 350, 300, 250, 200, 150, or 100 Dalton.

In various embodiments, the effector moiety has a molecular weight ofless than about 800 Dalton. For example, the effector moiety molecularweight can be less than about 800, 750, 700, 650, 600, 550, 500, 450,400, 350, 300, 250, 200, 150, or 100 Dalton.

In various embodiments, the binding moiety and the effector moiety areapproximately equal in size. For example, the binding moiety and theeffector moiety can have less than about a 25, 50, 75, 100, 125, 150,175, 200, 225, 250, 275, 300, 325, 350, 375, or 400 Dalton difference inmolecular weight.

In various embodiments, the binding moiety has a high affinity for amolecular target. For example, the binding moiety has a high affinityfor a molecular target that is a K_(d) of 50, 100, 150, 200, 250, 300,350, 400 nM or higher.

In various embodiments, when administered to a subject, the SDC-TRAP ispresent at a ratio of about 2:1, 5:1, 10:1, 25:1, 50:1, 75:1, 100:1,150:1, 200:1, 250:1, 300:1, 400:1, 500:1, 600:1, 700:1, 800:1, 900:1,1000:1, or greater. The ratio can be, for example, at 1, 2, 3, 4, 5, 6,7, 8, 12, 24, 48, 72, or more hours from administration.

In various embodiments, the SDC-TRAP is present in target cells and/ortissue for at least 24 hours. The SDC-TRAP can be present in cancercells for longer, for example, for at least 48, 72, 96, or 120 hours.

In various embodiments, the effector moiety is released for a period ofat least 6 hours. The effector moiety can be released for a longerperiod, for example, for at least 12, 24, 48, 72, 96, or 120 hours.

In various embodiments, the effector moiety is selectively releasedinside a target cell and/or tissue.

In various embodiments, the present invention provides SDC-TRAPmolecules comprising a binding moiety is an inhibitor of a targetprotein but that is ineffective as a therapeutic agent when administeredalone. In these, and in other embodiments, the SDC-TRAP may facilitatean additive or synergistic effect between the binding moiety andeffector moiety.

In various embodiments, the present invention provides method fortreating a subject having a cancer comprising administering atherapeutically effective amount of at least one SDC-TRAP to thesubject, thereby treating the cancer.

In various embodiments, the present invention provides a method fortreating a subject having a colon cancer comprising administering atherapeutically effective amount of at least one SDC-TRAP to thesubject, thereby treating the colon cancer.

In various embodiments, the present invention provides a method fortreating a subject having a breast cancer comprising administering atherapeutically effective amount of at least one SDC-TRAP to thesubject, thereby treating the breast cancer.

In various embodiments, the present invention provides a method fortreating a subject having an ovarian cancer comprising administering atherapeutically effective amount of at least one SDC-TRAP to thesubject, thereby treating the ovarian cancer.

In various embodiments, the present invention provides a method fortreating a subject having a lung cancer comprising administering atherapeutically effective amount of at least one SDC-TRAP to thesubject, thereby treating the lung cancer. The lung cancer can comprisesmall cell lung cancer.

In various embodiments, the present invention provides a method fortreating a subject having a skin cancer comprising administering atherapeutically effective amount of at least one SDC-TRAP to thesubject, thereby treating the skin cancer.

In various embodiments, the present invention provides a method fortreating a subject having actinic keratosis comprising administering atherapeutically effective amount of at least one SDC-TRAP to thesubject, thereby treating the actinic keratosis.

The present invention is described in further detail by the figures andexamples below, which are used only for illustration purposes and arenot limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows how an illustrative Hsp90-targeting moiety may be suitablymodified at one or more positions to enhance the physical,pharmacokinetic, or pharmacodynamic properties of the conjugate.

FIG. 2 illustrates an embodiment of a pharmaceutical conjugate havingtwo effector moieties. Other features and advantages of the instantinvention will be apparent from the following detailed description andclaims.

FIG. 3 is a table that shows a summary of the structure, physicalproperties, type of linkage, Her2 IC50 (nM) and cytotoxicity (BT474 IC50nM) for the exemplary SDC-TRAPs.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides molecules including an effector moietyconjugated to a binding moiety that directs the effector moiety to abiological target of interest. The molecules of the invention allow forselective targeting of an effector moiety by trapping the molecules ofthe invention in a desired cell, e.g., a cancer cell. The molecules canbe described as Small molecule Drug Congugates that are TRAPpedintracellularly (SDC-TRAP), due to their selective binding to highconcentration intracellular proteins. In order for the molecules of theinvention to be trapped within the cells of interest, the bindingmoieties that are part of the SDC-TRAP molecules interact with proteinsthat are overexpressed in targeted cells. In exemplary embodiments, theproteins that are overexpressed are characteristic of a particulardisease or disorder. Accordingly, the present invention providescompositions, kits, and methods (e.g., therapeutic, diagnostic, andimaging) that include the molecules of the invention.

In one embodiment of the invention, SDC-TRAPs allow for the delivery ofa effector molecule that would otherwise be unsuitable foradministration alone due to toxicity and/or undesired systemic effects.Using the targeted delivery molecules described herein (SDC-TRAPs)allows for effector moieties that are too toxic to administer by currentmethods to be dosed at lower levels thereby allowing the toxic effectorto be targeted to specific diseased cells at sub-toxic levels.

In various exemplary aspects and embodiments, the present inventionprovides compounds for treating cancer. For example, an SDC-TRAP cancomprise an Hsp90 binding moiety (i.e., targeting Hsp90, which isoverexpressed in cancer cells compared to normal cells) and an effectormoiety (e.g., the Hsp90 binding moiety can be an Hsp90 inhibitor that isconjugated to a cytotoxic agent). As indicated above, the invention isexemplified herein in terms of Hsp90-targeted binding moieties andcytotoxic agents. Other binding moieties that are contemplated,mentioned or described herein are intended to be included within thescope of the invention.

In various aspects and embodiments, the present invention provides anSDC-TRAP comprising a binding moiety and an effector moiety, wherein theSDC-TRAP molecule is able to enter a cell by passive transport. Theability of an SDC-TRAP to enter a cell by passive transport can be aresult of one or more unique chemical properties of the SDC-TRAP (e.g.,size, weight, charge, polarity, hydrophobicity, etc.) and can facilitatethe delivery and/or action of the SDC-TRAP. The ability of an SDC-TRAPto enter a cell by passive transport is a functional property, whichalong with its physico-chemical properties, differentiates SDC-TRAPsfrom other targeted molecules such as antibody-drug conjugates.

In various aspects and embodiments, the present invention provides anSDC-TRAP comprising a binding moiety and an effector moiety, whereinSDC-TRAP molecule is able to enter a cell by active transport. Theability of an SDC-TRAP to enter a cell by active transport can be aresult of one or more unique chemical properties of the SDC-TRAP and canfacilitate the delivery and/or action of the SDC-TRAP. Example ofSDC-TRAP active transport can include, for example, endocytosis,phagocytosis, pinocytosis, and exocytosis.

In various aspects and embodiments, the present invention provides anSDC-TRAP having a molecular weight of less than about 1600 Dalton (e.g.,less than about 1600, 1550, 1500, 1450, 1400, 1350, 1300, 1250, 1200,1150, 1100, 1050, 1000, 950, 900, 850, 800, 750, 700, 650, 600, 550,500, 450, 400, 350, 300, 250, 200, etc.). Similarly, in various aspectsand embodiments, the present invention provides a binding moiety havinga molecular weight of less than about 800 Dalton (e.g., less than about800, 750, 700, 650, 600, 550, 500, 450, 400, 350, 300, 250, 200, 150,100, etc.) and/or an effector moiety having a molecular weight of lessthan about 800 Dalton (e.g., less than about 800, 750, 700, 650, 600,550, 500, 450, 400, 350, 300, 250, 200, 150, 100, etc.). The overallmolecular weight of an SDC-TRAP, and the individual weights of a bindingmoiety, effector moiety, and any linking moiety, can affect transport ofthe SDC-TRAP. In various examples, it has been observed that lowermolecular weights can facilitate delivery and/or activity of anSDC-TRAP.

In various aspects and embodiments, the present invention provides anSDC-TRAP comprising an Hsp90 binding moiety and an effector moiety,wherein the Hsp90 binding moiety and the effector moiety areapproximately equal in size (e.g., the Hsp90 binding moiety and theeffector moiety have less than about a 25, 50, 75, 100, 125, 150, 175,200, 225, 250, 275, 300, 325, 350, 375, 400, etc. Dalton difference inmolecular weight.) In various examples, it has been observed that lowerdifferences in molecular weight can facilitate delivery and/or activityof an SDC-TRAP.

In various aspects and embodiments, the present invention provides anSDC-TRAP comprising a target protein-interacting binding moiety. Atarget protein-interacting binding moiety can selectively interact withany one or more domains of a target protein. For example, where a targetprotein is Hsp90, the binding moiety can be an Hsp90 binding moiety thatinteracts with the N-terminal domain of Hsp90, the C-terminal domain ofHsp90, and/or the middle domain of Hsp90. Selective interaction with anyone or more domains of a target protein can advantageously increasespecificity and/or increase the concentration of molecular targetswithin a target tissue and/or cell.

In various aspects and embodiments, the present invention provides anSDC-TRAP comprising a binding moiety having a high affinity for amolecular target (e.g., a K_(d) of 50, 100, 150, 200, 250, 300, 350, 400nM or higher). For example, where a binding moiety is an Hsp90 bindingmoiety, the Hsp90 binding moiety can have a K_(d) of 50, 100, 150, 200,250, 300, 350, 400 nM or higher. A binding moiety having a high affinityfor a molecular target can advantageously improve targeting and/orincrease the resonance time of the SDC-TRAP in a target cell and/ortissue.

In various aspects and embodiments, the present invention provides anSDC-TRAP comprising a binding moiety (e.g., Hsp90 binding moiety) and aneffector moiety, wherein when administered to a subject the SDC-TRAP ispresent at a ratio of about 2:1 in tumor cells compared to plasma. Theratio can be higher, for example, about 5:1, 10:1, 25:1, 50:1, 75:1,100:1, 150:1, 200:1, 250:1, 300:1, 400:1, 500:1, 600:1, 700:1, 800:1,900:1, 1000:1, or greater. In various aspects and embodiments, the ratiois at 1, 2, 3, 4, 5, 6, 7, 8, 12, 24, 48, 72, or more hours fromadministration. The effectiveness of targeting can be reflected in theratio of SDC-TRAP in a target cell and/or tissue compared to plasma.

In various aspects and embodiments, the present invention provides anSDC-TRAP comprising a binding moiety (e.g., Hsp90 binding moiety) and aneffector moiety, wherein the SDC-TRAP is present in target (e.g.,cancer) cells for at least 24 hours. The SDC-TRAP can be present incancer cells for longer, for example, for at least 48, 72, 96, or 120hours. It can be advantageous for an SDC-TRAP to be present in targetcells for longer periods of time to increase the therapeutic effect of agiven dose of SDC-TRAP and/or increase an interval betweenadministrations of SDC-TRAP.

In various aspects and embodiments, the present invention provides anSDC-TRAP comprising a binding moiety (e.g., Hsp90 binding moiety) and aneffector moiety, wherein the effector moiety is released for a period ofat least 6 hours. The effector moiety can be released for a longerperiod, for example, for at least 12, 24, 48, 72, 96, or 120 hours.Selective release can be used to control, delay, and/or extend theperiod of release of an effector moiety and, therefore, increase thetherapeutic effect of a given dose of SDC-TRAP, decrease the undesiredside effects of a given dose of SDC-TRAP, and/or increase an intervalbetween administrations of SDC-TRAP.

In various aspects and embodiments, the present invention provides anSDC-TRAP comprising an Hsp90 binding moiety and an effector moiety,wherein the effector moiety is selectively released inside a target(e.g., cancer) cell. Selective release can be achieved, for example, bya cleavable linker (e.g., an enzymatically cleavable linker). Selectiverelease can be used to decrease undesired toxicity and/or unwanted sideeffects. For example, an SDC-TRAP can be designed where an effectormoiety such is inactive (or relatively inactive) in a conjugated form,but active (or more active) after it is selectively released inside atarget (e.g., cancer) cell.

In various aspects and embodiments, the present invention provides anSDC-TRAP comprising a binding moiety (e.g., Hsp90 binding moiety) and aneffector moiety, wherein the SDC-TRAP allows for the use of an effectormoiety that is otherwise toxic or unfit for administration to a subject.The effector moiety can be unfit for administration to a subject becauseof undesired toxicity. In such cases, a strategy such as selectiverelease may be used to address the undesired toxicity. The effectormoiety can be unfit for administration to a subject because of undesiredtargeting or a lack of targeting. Targeting can address such problems,for example, by minimizing systemic toxicity while maximizing localtoxicity at a target (e.g., a tumor).

In various aspects and embodiments, the SDC-TRAP can exhibit decreasedand/or minimized toxicity concurrently with increased efficacy (e.g., ascompared to that of the effector moiety when used alone). Decreasingand/or minimizing toxicity can encompass reducing toxicity to apredetermined level (e.g., a regulatory guideline or suggested level,for example promulgated by the US Food and Drug Administration “FDA”).Increasing efficacy can encompass increasing efficacy to a predeterminedlevel (e.g., a regulatory guideline or suggested level, for examplepromulgated by the US FDA). Similarly, decreasing and/or minimizingtoxicity concurrently with increasing efficacy can encompass achieving apredetermined therapeutic ratio (e.g., a regulatory guideline orsuggested value, for example promulgated by the US FDA).

Decreasing and/or minimizing toxicity can encompass, for example,reducing toxicity by 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, 95%, or more. Increasing efficacy can encompass, forexample, increasing efficacy by 5, 10, 15, 20, 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 250, 300,400, 500%, or more. Decreasing and/or minimizing toxicity concurrentlywith increasing efficacy can encompass, for example: essentially thesame efficacy with decreased toxicity; essentially the same toxicitywith increased efficacy; or decreased toxicity and increased efficacy.Similarly, decreasing and/or minimizing toxicity concurrently withincreasing efficacy can encompass, for example, scenarios such as:increased efficacy enabling a lower dose (e.g., lower dose of effectormoiety with a correspondingly lower net toxicity) and decreased toxicityenabling a higher dose (e.g., higher dose of effector moiety without acorrespondingly higher net toxicity).

In various aspects and embodiments, the present invention provides anSDC-TRAP comprising a binding moiety (e.g., Hsp90 binding moiety) and aneffector moiety, wherein the binding moiety is an inhibitor (e.g., Hsp90inhibitor) that is ineffective as a therapeutic agent when administeredalone. In such cases, the SDC-TRAP may facilitate an additive orsynergistic effect between the binding moiety and effector moiety,thereby advantageously improving the efficacy and/or reducing the sideeffects of a therapy.

In order that the present invention may be more readily understood,certain terms are first defined. In addition, it should be noted thatwhenever a value or range of values of a parameter are recited, it isintended that values and ranges intermediate to the recited values arealso intended to be part of this invention. Unless defined otherwise,all technical and scientific terms used herein have the same meaning ascommonly understood to one of ordinary skill in the art to which thisinvention belongs. It is also to be understood that the terminologyemployed is for the purpose of describing particular embodiments, and isnot intended to be limiting.

DEFINITIONS

The articles “a,” “an,” and “the” are used herein to refer to one or tomore than one (i.e. to at least one) of the grammatical object of thearticle unless otherwise clearly indicated by contrast. By way ofexample, “an element” means one element or more than one element.

The term “including” is used herein to mean, and is used interchangeablywith, the phrase “including but not limited to.”

The term “or” is used herein to mean, and is used interchangeably with,the term “and/or,” unless context clearly indicates otherwise.

The term “such as” is used herein to mean, and is used interchangeably,with the phrase “such as but not limited to.”

Unless specifically stated or obvious from context, as used herein, theterm “about” is understood as within a range of normal tolerance in theart, for example within 2 standard deviations of the mean. About can beunderstood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%,0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear fromcontext, all numerical values provided herein can be modified by theterm about.

Ranges provided herein are understood to be shorthand for all of thevalues within the range. For example, a range of 1 to 50 is understoodto include any number, combination of numbers, or sub-range from thegroup consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

The recitation of a listing of chemical group(s) in any definition of avariable herein includes definitions of that variable as any singlegroup or combination of listed groups. The recitation of an embodimentfor a variable or aspect herein includes that embodiment as any singleembodiment or in combination with any other embodiments or portionsthereof.

Any compositions or methods provided herein can be combined with one ormore of any of the other compositions and methods provided herein.

As used herein, the term “subject” refers to human and non-humananimals, including veterinary subjects. The term “non-human animal”includes all vertebrates, e.g., mammals and non-mammals, such asnon-human primates, mice, rabbits, sheep, dog, cat, horse, cow,chickens, amphibians, and reptiles. In a preferred embodiment, thesubject is a human and may be referred to as a patient.

As used herein, the terms “treat,” “treating” or “treatment” refer,preferably, to an action to obtain a beneficial or desired clinicalresult including, but not limited to, alleviation or amelioration of oneor more signs or symptoms of a disease or condition, diminishing theextent of disease, stability (i.e., not worsening) state of disease,amelioration or palliation of the disease state, diminishing rate of ortime to progression, and remission (whether partial or total), whetherdetectable or undetectable. “Treatment” can also mean prolongingsurvival as compared to expected survival in the absence of treatment.Treatment does not need to be curative.

A “therapeutically effective amount” is that amount sufficient to treata disease in a subject. A therapeutically effective amount can beadministered in one or more administrations.

By “diagnosing” and the like, as used herein, refers to a clinical orother assessment of the condition of a subject based on observation,testing, or circumstances for identifying a subject having a disease,disorder, or condition based on the presence of at least one indicator,such as a sign or symptom of the disease, disorder, or condition.Typically, diagnosing using the method of the invention includes theobservation of the subject for multiple indicators of the disease,disorder, or condition in conjunction with the methods provided herein.Diagnostic methods provide an indicator that a disease is or is notpresent. A single diagnostic test typically does not provide adefinitive conclusion regarding the disease state of the subject beingtested.

The terms “administer,” “administering” or “administration” include anymethod of delivery of a pharmaceutical composition or agent into asubject's system or to a particular region in or on a subject. Incertain embodiments of the invention, an agent is administeredintravenously, intramuscularly, subcutaneously, intradermally,intranasally, orally, transcutaneously, or mucosally. In a preferredembodiment, an agent is administered intravenously. Administering anagent can be performed by a number of people working in concert.Administering an agent includes, for example, prescribing an agent to beadministered to a subject and/or providing instructions, directly orthrough another, to take a specific agent, either by self-delivery,e.g., as by oral delivery, subcutaneous delivery, intravenous deliverythrough a central line, etc.; or for delivery by a trained professional,e.g., intravenous delivery, intramuscular delivery, intratumoraldelivery, etc.

As used herein, the term “survival” refers to the continuation of lifeof a subject which has been treated for a disease or condition, e.g.,cancer. The time of survival can be defined from an arbitrary point suchas time of entry into a clinical trial, time from completion or failureor an earlier treatment regimen, time from diagnosis, etc.

As used herein, the term “recur” refers to the re-growth of tumor orcancerous cells in a subject in whom primary treatment for the tumor hasbeen administered. The tumor may recur in the original site or inanother part of the body. In one embodiment, a tumor that recurs is ofthe same type as the original tumor for which the subject was treated.For example, if a subject had an ovarian cancer tumor, was treated andsubsequently developed another ovarian cancer tumor, the tumor hasrecurred. In addition, a cancer can recur in or metastasize to adifferent organ or tissue than the one where it originally occurred.

As used herein, the terms “identify” or “select” refer to a choice inpreference to another. In other words, to identify a subject or select asubject is to perform the active step of picking out that particularsubject from a group and confirming the identity of the subject by nameor other distinguishing feature.

As used herein, the term “benefit” refers to something that isadvantageous or good, or an advantage. Similarly, the term “benefiting,”as used herein, refers to something that improves or advantages. Forexample, a subject will benefit from treatment if they exhibit adecrease in at least one sign or symptom of a disease or condition(e.g., tumor shrinkage, decrease in tumor burden, inhibition or decreaseof metastasis, improving quality of life (“QOL”), if there is a delay oftime to progression (“TTP”), if there is an increase of overall survival(“OS”), etc.), or if there is a slowing or stopping of diseaseprogression (e.g., halting tumor growth or metastasis, or slowing therate of tumor growth or metastasis). A benefit can also include animprovement in quality of life, or an increase in survival time orprogression free survival.

The terms “cancer” or “tumor” are well known in the art and refer to thepresence, e.g., in a subject, of cells possessing characteristicstypical of cancer-causing cells, such as uncontrolled proliferation,immortality, metastatic potential, rapid growth and proliferation rate,decreased cell death/apoptosis, and certain characteristic morphologicalfeatures. Cancer cells are often in the form of a solid tumor. However,cancer also includes non-solid tumors, e.g., blood tumors, e.g.,leukemia, wherein the cancer cells are derived from bone marrow. As usedherein, the term “cancer” includes pre-malignant as well as malignantcancers. Cancers include, but are not limited to, acoustic neuroma,acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia(monocytic, myeloblastic, adenocarcinoma, angiosarcoma, astrocytoma,myelomonocytic and promyelocytic), acute T-cell leukemia, basal cellcarcinoma, bile duct carcinoma, bladder cancer, brain cancer, breastcancer, bronchogenic carcinoma, cervical cancer, chondrosarcoma,chordoma, choriocarcinoma, chronic leukemia, chronic lymphocyticleukemia, chronic myelocytic (granulocytic) leukemia, chronicmyelogenous leukemia, colon cancer, colorectal cancer,craniopharyngioma, cystadenocarcinoma, diffuse large B-cell lymphoma,Burkitt's lymphoma, dysproliferative changes (dysplasias andmetaplasias), embryonal carcinoma, endometrial cancer,endotheliosarcoma, ependymoma, epithelial carcinoma, erythroleukemia,esophageal cancer, estrogen-receptor positive breast cancer, essentialthrombocythemia, Ewing's tumor, fibrosarcoma, follicular lymphoma, germcell testicular cancer, glioma, heavy chain disease, hemangioblastoma,hepatoma, hepatocellular cancer, hormone insensitive prostate cancer,leiomyosarcoma, liposarcoma, lung cancer, lymphagioendotheliosarcoma,lymphangiosarcoma, lymphoblastic leukemia, lymphoma (Hodgkin's andnon-Hodgkin's), malignancies and hyperproliferative disorders of thebladder, breast, colon, lung, ovaries, pancreas, prostate, skin, anduterus, lymphoid malignancies of T-cell or B-cell origin, leukemia,lymphoma, medullary carcinoma, medulloblastoma, melanoma, meningioma,mesothelioma, multiple myeloma, myelogenous leukemia, myeloma,myxosarcoma, neuroblastoma, non-small cell lung cancer,oligodendroglioma, oral cancer, osteogenic sarcoma, ovarian cancer,pancreatic cancer, papillary adenocarcinomas, papillary carcinoma,pinealoma, polycythemia vera, prostate cancer, rectal cancer, renal cellcarcinoma, retinoblastoma, rhabdomyosarcoma, sarcoma, sebaceous glandcarcinoma, seminoma, skin cancer, small cell lung carcinoma, solidtumors (carcinomas and sarcomas), small cell lung cancer, stomachcancer, squamous cell carcinoma, synovioma, sweat gland carcinoma,thyroid cancer, Waldenstrom's macroglobulinemia, testicular tumors,uterine cancer, and Wilms' tumor. Other cancers include primary cancer,metastatic cancer, oropharyngeal cancer, hypopharyngeal cancer, livercancer, gall bladder cancer, bile duct cancer, small intestine cancer,urinary tract cancer, kidney cancer, urothelium cancer, female genitaltract cancer, uterine cancer, gestational trophoblastic disease, malegenital tract cancer, seminal vesicle cancer, testicular cancer, germcell tumors, endocrine gland tumors, thyroid cancer, adrenal cancer,pituitary gland cancer, hemangioma, sarcoma arising from bone and softtissues, Kaposi's sarcoma, nerve cancer, ocular cancer, meningialcancer, glioblastomas, neuromas, neuroblastomas, Schwannomas, solidtumors arising from hematopoietic malignancies such as leukemias,metastatic melanoma, recurrent or persistent ovarian epithelial cancer,fallopian tube cancer, primary peritoneal cancer, gastrointestinalstromal tumors, colorectal cancer, gastric cancer, melanoma,glioblastoma multiforme, non-squamous non-small-cell lung cancer,malignant glioma, epithelial ovarian cancer, primary peritoneal serouscancer, metastatic liver cancer, neuroendocrine carcinoma, refractorymalignancy, triple negative breast cancer, HER2-amplified breast cancer,nasopharageal cancer, oral cancer, biliary tract, hepatocellularcarcinoma, squamous cell carcinomas of the head and neck (SCCHN),non-medullary thyroid carcinoma, recurrent glioblastoma multiforme,neurofibromatosis type 1, CNS cancer, liposarcoma, leiomyosarcoma,salivary gland cancer, mucosal melanoma, acral/lentiginous melanoma,paraganglioma, pheochromocytoma, advanced metastatic cancer, solidtumor, triple negative breast cancer, colorectal cancer, sarcoma,melanoma, renal carcinoma, endometrial cancer, thyroid cancer,rhabdomysarcoma, multiple myeloma, ovarian cancer, glioblastoma,gastrointestinal stromal tumor, mantle cell lymphoma, and refractorymalignancy.

“Solid tumor,” as used herein, is understood as any pathogenic tumorthat can be palpated or detected using imaging methods as an abnormalgrowth having three dimensions. A solid tumor is differentiated from ablood tumor such as leukemia. However, cells of a blood tumor arederived from bone marrow; therefore, the tissue producing the cancercells is a solid tissue that can be hypoxic.

“Tumor tissue” is understood as cells, extracellular matrix, and othernaturally occurring components associated with the solid tumor.

As used herein, the term “isolated” refers to a preparation that issubstantially free (e.g., 50%, 60%, 70%, 80%, 90% or more, by weight)from other proteins, nucleic acids, or compounds associated with thetissue from which the preparation is obtained.

The term “sample” as used herein refers to a collection of similarfluids, cells, or tissues isolated from a subject. The term “sample”includes any body fluid (e.g., urine, serum, blood fluids, lymph,gynecological fluids, cystic fluid, ascetic fluid, ocular fluids, andfluids collected by bronchial lavage and/or peritoneal rinsing),ascites, tissue samples (e.g., tumor samples) or a cell from a subject.Other subject samples include tear drops, serum, cerebrospinal fluid,feces, sputum, and cell extracts. In one embodiment, the sample isremoved from the subject. In a particular embodiment, the sample isurine or serum. In another embodiment, the sample does not includeascites or is not an ascites sample. In another embodiment, the sampledoes not include peritoneal fluid or is not peritoneal fluid. In oneembodiment, the sample comprises cells. In another embodiment, thesample does not comprise cells. Samples are typically removed from thesubject prior to analysis. However, tumor samples can be analyzed in thesubject, for example, using imaging or other detection methods.

The term “control sample,” as used herein, refers to any clinicallyrelevant comparative sample, including, for example, a sample from ahealthy subject not afflicted with cancer, a sample from a subjecthaving a less severe or slower progressing cancer than the subject to beassessed, a sample from a subject having some other type of cancer ordisease, a sample from a subject prior to treatment, a sample ofnon-diseased tissue (e.g., non-tumor tissue), a sample from the sameorigin and close to the tumor site, and the like. A control sample canbe a purified sample, protein, and/or nucleic acid provided with a kit.Such control samples can be diluted, for example, in a dilution seriesto allow for quantitative measurement of analytes in test samples. Acontrol sample may include a sample derived from one or more subjects. Acontrol sample may also be a sample made at an earlier time point fromthe subject to be assessed. For example, the control sample could be asample taken from the subject to be assessed before the onset of thecancer, at an earlier stage of disease, or before the administration oftreatment or of a portion of treatment. The control sample may also be asample from an animal model, or from a tissue or cell lines derived fromthe animal model, of the cancer. The level in a control sample thatconsists of a group of measurements may be determined, e.g., based onany appropriate statistical measure, such as, for example, measures ofcentral tendency including average, median, or modal values.

As used herein, the term “obtaining” is understood herein asmanufacturing, purchasing, or otherwise coming into possession of.

As used herein, the term “identical” or “identity” is used herein inrelation to amino acid or nucleic acid sequences refers to any gene orprotein sequence that bears at least 30% identity, more preferably 40%,50%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, and most preferably 95%, 96%, 97%, 98%, 99% ormore identity to a known gene or protein sequence over the length of thecomparison sequence. Protein or nucleic acid sequences with high levelsof identity throughout the sequence can be said to be homologous. A“homologous” protein can also have at least one biological activity ofthe comparison protein. In general, for proteins, the length ofcomparison sequences will be at least 10 amino acids, preferably 10, 20,30, 40, 50, 60, 70, 80, 90, 100, 150, 175, 200, 250, or at least 300amino acids or more. For nucleic acids, the length of comparisonsequences will generally be at least 25, 50, 100, 125, 150, 200, 250,300, 350, 400, 450, 500, 550, 600, 650, 700, 800, or at least 850nucleotides or more.

As used herein, “detecting,” “detection” and the like are understoodthat an assay performed for identification of a specific analyte in asample. The amount of analyte or activity detected in the sample can benone or below the level of detection of the assay or method.

The terms “modulate” or “modulation” refer to upregulation (i.e.,activation or stimulation), downregulation (i.e., inhibition orsuppression) of a level, or the two in combination or apart. A“modulator” is a compound or molecule that modulates, and may be, e.g.,an agonist, antagonist, activator, stimulator, suppressor, or inhibitor.

The term “expression” is used herein to mean the process by which apolypeptide is produced from DNA. The process involves the transcriptionof the gene into mRNA and the translation of this mRNA into apolypeptide. Depending on the context in which used, “expression” mayrefer to the production of RNA, or protein, or both.

The terms “level of expression of a gene” or “gene expression level”refer to the level of mRNA, as well as pre-mRNA nascent transcript(s),transcript processing intermediates, mature mRNA(s) and degradationproducts, or the level of protein, encoded by the gene in the cell.

As used herein, “level of activity” is understood as the amount ofprotein activity, typically enzymatic activity, as determined by aquantitative, semi-quantitative, or qualitative assay. Activity istypically determined by monitoring the amount of product produced in anassay using a substrate that produces a readily detectable product,e.g., colored product, fluorescent product, or radioactive product.

As used herein, “changed as compared to a control” sample or subject isunderstood as having a level of the analyte or diagnostic or therapeuticindicator (e.g., marker) to be detected at a level that is statisticallydifferent than a sample from a normal, untreated, or control samplecontrol samples include, for example, cells in culture, one or morelaboratory test animals, or one or more human subjects. Methods toselect and test control samples are within the ability of those in theart. An analyte can be a naturally occurring substance that ischaracteristically expressed or produced by the cell or organism (e.g.,an antibody, a protein) or a substance produced by a reporter construct(e.g., β-galactosidase or luciferase). Depending on the method used fordetection the amount and measurement of the change can vary. Changed ascompared to a control reference sample can also include a change in oneor more signs or symptoms associated with or diagnostic of disease,e.g., cancer. Determination of statistical significance is within theability of those skilled in the art, e.g., the number of standarddeviations from the mean that constitute a positive result.

“Elevated” or “lower” refers to a patient's value of a marker relativeto the upper limit of normal (“ULN”) or the lower limit of normal(“LLN”) which are based on historical normal control samples. As thelevel of the marker present in the subject will be a result of thedisease, and not a result of treatment, typically a control sampleobtained from the patient prior to onset of the disease will not likelybe available. Because different labs may have different absoluteresults, values are presented relative to that lab's upper limit ofnormal value (ULN).

The “normal” level of expression of a marker is the level of expressionof the marker in cells of a subject or patient not afflicted withcancer. In one embodiment, a “normal” level of expression refers to thelevel of expression of the marker under normoxic conditions.

An “over-expression” or “high level of expression” of a marker refers toan expression level in a test sample that is greater than the standarderror of the assay employed to assess expression, and is preferably atleast 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3,2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 4, 5, 6, 7, 8, 9, or 10 times theexpression level of the marker in a control sample (e.g., sample from ahealthy subject not having the marker associated disease, i.e., cancer).In one embodiment, expression of a marker is compared to an averageexpression level of the marker in several control samples.

A “low level of expression” or “under-expression” of a marker refers toan expression level in a test sample that is less than at least 0.9,0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0. 1 times the expression level ofthe marker in a control sample (e.g., sample from a healthy subject nothaving the marker associated disease, i.e., cancer). In one embodiment,expression of a marker is compared to an average expression level of themarker in several control samples.

As used herein, “binding” is understood as having at least a 10² ormore, 10³ or more, preferably 10⁴ or more, preferably 10⁵ or more,preferably 10⁶ or more preference for binding to a specific bindingpartner as compared to a non-specific binding partner (e.g., binding anantigen to a sample known to contain the cognate antibody).

“Determining” as used herein is understood as performing an assay orusing a diagnostic method to ascertain the state of someone orsomething, e.g., the presence, absence, level, or degree of a certaincondition, biomarker, disease state, or physiological condition.

“Prescribing” as used herein is understood as indicating a specificagent or agents for administration to a subject.

As used herein, the terms “respond” or “response” are understood ashaving a positive response to treatment with a therapeutic agent,wherein a positive response is understood as having a decrease in atleast one sign or symptom of a disease or condition (e.g., tumorshrinkage, decrease in tumor burden, inhibition or decrease ofmetastasis, improving quality of life (“QOL”), delay of time toprogression (“TTP”), increase of overall survival (“OS”), etc.), orslowing or stopping of disease progression (e.g., halting tumor growthor metastasis, or slowing the rate of tumor growth or metastasis). Aresponse can also include an improvement in quality of life, or anincrease in survival time or progression free survival.

The terms “administer,” “administering” or “administration” can includeany method of delivery of a pharmaceutical composition or agent into asubject's system or to a particular region in or on a subject. Incertain embodiments of the invention, an Hsp90 inhibitor is administeredintravenously, intramuscularly, subcutaneously, intradermally,intranasally, orally, transcutaneously, or mucosally. In a preferredembodiment, an agent is administered intravenously. Administering can beperformed by a number of people working in concert. Administering anagent includes, for example, prescribing an agent to be administered toa subject and/or providing instructions, directly or through another, totake a specific agent, either by self-delivery, e.g., as by oraldelivery, subcutaneous delivery, intravenous delivery through a centralline, etc.; or for delivery by a trained professional, e.g., intravenousdelivery, intramuscular delivery, intratumoral delivery, etc.

As used herein, the term “high concentration” refers to theconcentration of SDC-TRAP that accumulates in target cells of theinvention due to the selective binding of the binding moiety of theSDC-TRAP to the target protein. In one embodiment, the concentration ishigher than in similar cells that do not overexpress the target protein,e.g., lung cancer cells as compared to non-cancerous lung cells. Inanother embodiment, the concentration is higher in target cells comparedto cells that do not express, or overexpress, the target protein. Inexemplary embodiments, the high concentration is 1.5, 2, 3, 4, 5, 10,15, 20, 50, 100, 1000 times or more than cells that are not targeted bythe SDC-TRAP molecules of the invention.

The term “moiety” refers generally to a portion of a molecule, which maybe a functional group, a set of functional groups, and/or a specificgroup of atoms within a molecule, that is responsible for acharacteristic chemical, biological, and/or medicinal property of themolecule.

The term “binding moiety” refers to low molecular weight (e.g., lessthan about 800, 700, 600, 500, 400, 300, 200, or 100 etc. Dalton)organic compounds, which may serve as a therapeutic or a regulator of abiological process. Binding moieties include molecules that can bind toa biopolymer such as protein, nucleic acid, or polysaccharide and actsas an effector, altering the activity or function of the biopolymer.Binding moieties can have a variety of biological functions, serving ascell signaling molecules, as tools in molecular biology, as drugs inmedicine, as pesticides in farming, and in many other roles. Thesecompounds can be natural (such as secondary metabolites) or artificial(such as antiviral drugs); they may have a beneficial effect against adisease (such as drugs) or may be detrimental (such as teratogens andcarcinogens). Biopolymers such as nucleic acids, proteins, andpolysaccharides (such as starch or cellulose) are not binding moieties,although their constituent monomers—ribo- or deoxyribo-nucleotides,amino acids, and monosaccharides, respectively—are often considered tobe. Small oligomers are also usually considered binding moieties, suchas dinucleotides, peptides such as the antioxidant glutathione, anddisaccharides such as sucrose.

As used herein, a “protein interacting binding moiety” or “bindingmoiety” refers to a binding moiety, or portion thereof, that interactswith a predetermined target. The interaction is achieved through somedegree of specificity and/or affinity for the target. Both specificityand affinity is generally desirable, although in certain cases higherspecificity may compensate for lower affinity and higher affinity maycompensate for lower specificity. Affinity and specificity requirementswill vary depending upon various factors including, but not limited to,absolute concentration of the target, relative concentration of thetarget (e.g., in cancer vs. normal cells), potency and toxicity, routeof administration, and/or diffusion or transport into a target cell. Thetarget can be a molecule of interest and/or localized in an area ofinterest. For example, the target can be a therapeutic target and/orlocalized in an area targeted for a therapy (e.g., a protein that isoverexpressed in cancerous cells, as compared to normal cells). In oneparticular example, a target can be a chaperonin protein such as Hsp90and the binding moiety can be an Hsp90 binding moiety (e.g.,therapeutic, cytotoxic, or imaging moiety). Preferentially, the bindingmoiety will enhance, be compatible with, or not substantially reduce,passive transport of a conjugate including the binding moiety into acell, e.g., a cell comprising a target protein.

The term “effector moiety” refers to a molecule, or portion thereof,that has an effect on a target and/or proximally to the target. Invarious preferred embodiments, the effector moiety is a binding moiety,or portion thereof. An effect can include, but is not limited to, atherapeutic effect, an imaging effect, and/or a cytotoxic effect. At amolecular or cellular level, an effect can include, but is not limitedto, promotion or inhibition of the target's activity, labeling of thetarget, and/or cell death. Preferentially, the effector moiety willenhance, be compatible with, or not substantially reduce, passivetransport of a conjugate including the effector moiety into a cellcomprising a target. Different effector moieties can be used togetherand therapeutics in accordance with the present invention may includemore than one effector moiety (e.g., two or more different (or same)effector moieties in a single therapeutic in accordance with the presentinvention, two or more different therapeutics in accordance with thepresent invention including different effector moieties).

In some embodiments, the effector moiety is selected from the groupconsisting of LY2801653, LY2835219, LY2606368, LY2874455, LY2090314,LY2940680, LY278544, LY2228820, LY2157299, LY2603618, LY353381, andtrimetrexate.

The term “small molecule drug conjugate that is trapped intracellularly”or “binding moiety drug conjugate that is trapped intracellularly” or“SDC-TRAP” refers to a binding moiety and effector moiety joined to oneanother, or acting as if joined to one another. A binding moiety andeffector moiety can be joined through essentially any chemical orphysical force, either directly (e.g., binding moiety and effectormoiety viewed as two moieties on the same molecule, or a single moietyhaving both functions) or through an intermediate (e.g., linker). Forexample, a binding moiety and effector moiety can be joined by one ormore covalent bonds, ionic bonds, hydrogen bonds, the hydrophobiceffect, dipole-dipole forces, ion-dipole forces, dipole-induced dipoleforces, instantaneous dipole-induced dipole forces, and/or combinationsthereof. Preferentially, the SDC-TRAP will be capable of passive and/oractive transport into a cell comprising a target. Moreover, SDC-TRAPmolecules of the invention may comprise multiple effector moleculesconjugated to the binding moiety.

The term “linker” or “linking moiety,” as used herein in the context ofbinding moiety, effector moieties, and/or SDC-TRAPs refers to a chemicalmoiety that joins two other moieties (e.g., a binding moiety and aneffector moiety). A linker can covalently join a binding moiety and aneffector moiety. A linker can include a cleavable linker, for example anenzymatically cleavable linker. A linker can include a disulfide,carbamate, amide, ester, and/or ether linkers.

In some embodiments, the linker or linking moiety of an SDC-TRAP can beadvantageous when compared to the limited linking chemistry ofantibody-drug conjugates (ADC). For example, unlike ADCs that arelimited by the need to maintain the structure and/or stability of anantibody, SDC-TRAPs can use a wider range of linking chemistries and/orsolvents (e.g., that can alter, distort, or denature an antibody).

As used herein, a “ligand” is a substance (e.g., a binding moiety) thatcan form a complex with a biomolecule. The ligand and/or formation ofthe ligand-biomolecule complex can have a biological or chemical effect,such as a therapeutic effect, cytotoxic effect, and/or imaging effect.

As used herein, a “prodrug” is a pharmacological substance that isadministered in an inactive or less than fully active form and that issubsequently converted to an active pharmacological agent (i.e., thedrug) through a metabolic processes. Prodrugs can be used to improve howthe intended drug is absorbed, distributed, metabolized, and/orexcreted. A prodrug may also be used to improve how selectively theintended drug interacts with cells or processes that are not itsintended target (e.g., to reduce adverse or unintended effects of theintended drug, for example a chemotherapy drug).

The phrase “Hsp90 ligand or a prodrug thereof” refers generally tomolecules that bind to and in some cases effect Hsp90, and inactiveforms (i.e., prodrugs) thereof. An Hsp90 ligand can be an “Hsp90inhibitor,” which is understood as a therapeutic agent that reduces theactivity of Hsp90 either by directly interacting with Hsp90 or by, forexample, preventing the formation of the Hsp90/CDC37 complex such thatthe expression and proper folding of at least one client protein ofHsp90 is inhibited. “Hsp90” includes each member of the family of heatshock proteins having a mass of about 90-kilodaltons. For example, inhumans the highly conserved Hsp90 family includes cytosolic Hsp90^(α)and Hsp90^(β) isoforms, as well as GRP94, which is found in theendoplasmic reticulum, and HSP75/TRAP1, which is found in themitochondrial matrix. As used herein, Hsp90 inhibitors include, but arenot limited to ganetespib, geldanamycin (tanespimycin), e.g., IPI-493,macbecins, tripterins, tanespimycins, e.g., 17-AAG (alvespimycin),KF-55823, radicicols, KF-58333, KF-58332, 17-DMAG, IPI-504, BIIB-021,BIIB-028, PU-H64, PU-H71, PU-DZ8, PU-HZ151, SNX-2112, SNX-2321,SNX-5422, SNX-7081, SNX-8891, SNX-0723, SAR-567530, ABI-287, ABI-328,AT-13387, NSC-113497, PF-3823863, PF-4470296, EC-102, EC-154,ARQ-250-RP, BC-274, VER-50589, KW-2478, BHI-001, AUY-922, EMD-614684,EMD-683671, XL-888, VER-51047, KOS-2484, KOS-2539, CUDC-305, MPC-3100,CH-5164840, PU-DZ13, PU-HZ151, PU-DZ13, VER-82576, VER-82160, VER-82576,VER-82160, NXD-30001, NVP-HSP990, SST-0201CL1, SST-0115AA1, SST-0221AA1,SST-0223AA1, novobiocin (a C-terminal Hsp90i, herbinmycin A, radicicol,CCT018059, PU-H71, or celastrol.

The term “therapeutic moiety” refers to molecule, compound, or fragmentthereof that is used for the treatment of a disease or for improving thewell-being of an organism or that otherwise exhibit healing power (e.g.,pharmaceuticals, drugs, and the like). A therapeutic moiety can be achemical, or fragment thereof, of natural or synthetic origin used forits specific action against disease, for example cancer. Therapeuticagents used for treating cancer may be called chemotherapeutic agents.As described herein, a therapeutic moiety is preferentially a smallmolecule. Exemplary small molecule therapeutics include those that areless than 800 Daltons, 700 Daltons, 600 Daltons, 500 Daltons, 400Daltons, or 300 Daltons.

The term “cytotoxic moiety” refers to molecule, compound, or fragmentthereof that has a toxic or poisonous effect on cells, or that killscells. Chemotherapy and radiotherapy are forms of cytotoxic therapy.Treating cells with a cytotoxic moiety can produce a variety ofresults—cells may undergo necrosis, stop actively growing and dividing,or activate a genetic program of controlled cell death (i.e.,apoptosis). Examples of cytotoxic moieties include, but are not limitedto, SN-38, bendamustine, VDA, doxorubicin, pemetrexed, vorinostat,lenalidomide, irinotecan, ganetespib, docetaxel, 17-AAG, 5-FU,abiraterone, crizotinib, KW-2189, BUMB2, DC1, CC-1065, adozelesin, orfragment(s) thereof.

The term “imaging moiety” refers to a molecule, compound, or fragmentthereof that facilitates a technique and/or process used to createimages or take measurements of a cell, tissue, and/or organism (or partsor functions thereof) for clinical and/or research purposes. An imagingmoiety can produce, for example, a signal through emission and/orinteraction with electromagnetic, nuclear, and/or mechanical (e.g.,acoustic as in ultrasound) energy. An imaging moiety can be used, forexample, in various radiology, nuclear medicine, endoscopy,thermography, photography, spectroscopy, and microscopy methods.

“Pharmaceutical conjugate” refers to a non-naturally occurring moleculethat includes a binding moiety (e.g., an Hsp90-targeting moiety)associated with an effector moiety, where these two components may alsobe covalently bonded to each other either directly or through a linkinggroup.

The term “drug” refers to any active agent that affects any biologicalprocess. Active agents that are considered drugs for purposes of thisapplication are agents that exhibit a pharmacological activity. Examplesof drugs include active agents that are used in the prevention,diagnosis, alleviation, treatment or cure of a disease condition.

By “pharmacologic activity” is meant an activity that modulates oralters a biological process so as to result in a phenotypic change,e.g., cell death, cell proliferation etc.

By “pharmacokinetic property” is meant a parameter that describes thedisposition of an active agent in an organism or host.

By “half-life” is meant the time for one-half of an administered drug tobe eliminated through biological processes, e.g., metabolism, excretion,etc.

The term “efficacy” refers to the effectiveness of a particular activeagent for its intended purpose, i.e., the ability of a given activeagent to cause its desired pharmacologic effect.

Binding Moiety-Effector Moiety Drug Conjugates that are TrappedIntracellularly (SDC-TRAPs)

The present invention provides SDC-TRAPs, as well as SDC-TRAPcompositions, kits, and methods of use thereof. SDC-TRAPs include abinding moiety (e.g., a binding moiety such as a ligand) conjugated toan effector moiety (e.g., a pharmacological agent such as a drug orimaging agent). These two moieties can be joined by a linker, e.g., acovalently-bonded linking group. SDC-TRAPs are useful in a variety oftherapeutic, imaging, diagnostic, and/or research applications. In oneillustrative example of cancer therapy, an SDC-TRAP can be apharmaceutical conjugate of an Hsp90-binding moiety such as an Hsp90ligand or inhibitor associated with an effector moiety such as atherapeutic or cytotoxic agent.

In various embodiments, an SDC-TRAP can be further characterized in thatthe binding moiety (e.g., targeting moiety) and effector moiety aredifferent, such that the pharmaceutical conjugate may be viewed as aheterodimeric compound produced by the joining of two differentmoieties. In terms of function, SDC-TRAP molecules have a targetingfunctionality and effector functionality (e.g., therapeutic, imaging,diagnostic). These functions are provided by corresponding chemicalmoieties that can be different (or, in some cases, the same). SDC-TRAPscan include any one or more binding moieties conjugated to any one ormore effector moieties. In some embodiments, a composition or method caninclude a combination of two or more binding moeities and/or two or moreeffector moieties (e.g., a combination therapy and/or multi targettherapy) embodied in one or more different types of SDC-TRAPs.

In various embodiments, an SDC-TRAP is further characterized by itsability to passively diffuse and/or be actively transported into atarget cell of interest. The diffusion and/or transport properties ofthe SDC-TRAP can be derived, at least in part, from ionic, polar, and/orhydrophobic properties of the SDC-TRAP. In preferred embodiments, theSDC-TRAP enter cells primarily by passive diffusion. The diffusionand/or transport properties of the SDC-TRAP can be derived, at least inpart, from the molecular weight of the SDC-TRAP, the binding moiety, theeffector moiety, and/or the similarity in weight between the bindingmoiety and the effector moiety. SDC-TRAPs are desirably small, such asin comparison to antibody-drug conjugates (“ADCs”). For example, themolecular weight of an SDC-TRAP can be less than about 1600, 1500, 1400,1300, 1200, 1100, 1000, 900, 800, 700, 600, 500, or 400 Daltons. Abinding moiety and an effector moiety can each be less than about 1000,900, 800, 700, 600, 500, 400, 300, or 200 Daltons. A binding moiety andan effector moiety can be approximately equal in size (e.g., differ inweight by less than 400, 350, 300, 250, 200, 150, 100, or 50 Daltons).

Delivery of an effector molecule by an SDC-TRAP can result in greaterpotency compared to administering an untargeted drug comprising the sameeffector moiety, for example, because the SDC-TRAP can be localized at adesired target for an extended period of time through the association ofa binding moiety and its target. Such localization can cause an effectormoiety to be active and/or released in a target cell and/or tissue overan extended period of time. This resonance time can be selected throughdeliberate design of a linker moiety. In contrast, administration of thedrug by itself in vivo can be more apt to have a shorter resonance timein a given target cell and/or tissue—if it traverses into the cell atall—due to the lack of an “anchor” within the cell.

SDC-TRAPs, in part because they comprise a targeting moiety and arerelatively small in size, can be efficiently taken up or internalized bya target cell. Conversely, uptake or internalization is relativelyinefficient for ADCs, which must deal with limited antigen expressionand relatively inefficient internalization mechanisms for the antibodyportion of the molecule. Hsp90 provides a good illustrative example of adifference between SDC-TRAPs and conventional ADCs. By way ofcomparison, the localization rate of radiolabeled monoclonal antibodiesat a tumor in patients is low, on the order of 0.003-0.08% of theinjected dose/g tumor. In contrast, a much higher accumulation rate(15-20% injected dose/g tumor) has been measured for SDC-TRAPs in mousetumor xenografts.

SDC-TRAP pharmaceutical conjugates in accordance with the presentinvention can represent a significant advance over the state of the artin targeted drugs. SDC-TRAPs have broad application in many therapeutic,imaging, and diagnostic application. As discussed above, SDC-TRAPs areadvantageously small in comparison to ADCs, enabling better penetrationof solid tumors and more rapid clearance from normal tissues (e.g.,reduced toxicity). The design of SDC-TRAPs (e.g., a structure-propertyrelationship) can be established using methods and rationales within thegrasp of those of ordinary skill in the art, and companion imagingdiagnostics for targeted therapies may also easily be provided, in viewof the simpler chemistry involved.

SDC-TRAPs of the invention are characterized by selective targeting ofSDC-TRAPs to target cells in which a target protein is overexpressed.This leads to high intracellular concentrations of SDC-TRAP molecules intarget cells as compared to non-targeted cells. Likewise, SDC-TRAPs ofthe invention are characterized by low concentrations of SDC-TRAP innon-targeted cells.

One illustrative embodiment involves a conjugate of an Hsp90 bindingmoiety linked to a chelator (i.e., the effector moiety, for metals suchas In or Gd, which conjugate may function as an imaging agent for thecells/tissues targeted by the conjugate). Another, illustrativeembodiment involves a conjugate of an Hsp90 binding moiety linked to achemotherapeutic (i.e., the effector moiety, for example, SN-38).Alternatively, an illustrative SDC-TRAP is contemplated wherein an Hsp90targeting moiety bearing radiolabeled halogen (e.g., such as an iodineisotope) can serve to image the cells/tissues targeted by the conjugate,and the effector moiety can be drug to treat the targeted cells/tissues.The progression of treatment may therefore be determined by imaging thetissues being treated and reviewing the images for the presence orabsence of the labeled conjugate. Such embodiments are readily adaptableto essentially any cancer, or other chemotherapeutic target. Moleculartargets (e.g., interacting with a binding moiety) used to target aparticular cell or tissue can be selected based upon their presence inthe target cell or tissue and/or their relative abundance in the targetcell or tissue (e.g., disease-related versus normal cells).

SDC-TRAP molecules of the present invention represent a new class ofdrugs. One particular advantage of SDC-TRAPs is that they can bedesigned to selectively deliver an effector moiety (e.g., achemotherapeutic drug) into a targeted cell because of the relativeoverexpression or presence of a binding moiety's molecular target in thecell. After the binding moiety binds the molecular target, the effectormoiety is thereafter available (e.g., through cleavage of a linkermoiety joining the binding moiety and the effector moiety) to act uponthe cell. Accordingly, SDC-TRAPs employ a different mechanism fromstrategies currently used in the art, for example delivering an Hsp90inhibitor to a cell using HPMA copolymer-Hsp90i conjugates, Hsp90iprodrugs, nanoparticle-Hsp90i conjugates, or micellar methodologies.

SDC-TRAPs can also described by the formula:

Binding moiety-L-E

Where “binding moiety” is a protein interacting binding moiety; L is aconjugation or linking moiety (e.g., a bond or a linking group); and Eis an effector moiety. These elements are discussed in the context ofadditional illustrative examples below. However, while features of eachelement may be discussed separately, design and selection of an SDC-TRAPcan involve the interplay and/or cumulative effect of features of eachelement (e.g., diffusion, binding, and effect).

Once SDC-TRAP molecules of the invention enter a target cell theeffector molecule is released from the SDC-TRAP. In one embodiment, theeffector molecule has no activity until it is released from theSDC-TRAP. Accordingly, once the SDC-TRAP molecules enter a target cellan equilibrium exists between free and bound SDC-TRAP molecules. In oneembodiment, the effector moiety is only released from the SDC-TRAP whenthe SDC-TRAP is not associated with the target protein. For example,when an SDC-TRAP molecule is not bound intracellular enzymes can accessthe linker region thereby freeing the effector moiety. Alternatively,when free SDC-TRAP molecules may be able to release effector moleculesthrough, for example, hydrolysis of the bond or linker that connects thebinding moiety and effector moiety.

Accordingly, the rate of effector molecule release and the amount ofeffector molecule released can be controlled by using binding moietiesthat bind to the target protein with different affinities. For example,binding moieties that bind to the target protein with lower affinitywill be free, resulting in higher concentrations of unboundintracellular SDC-TRAP, and thereby resulting in higher concentrationsof free effector molecule. Therefore, in at least one embodiment,irreversibly-binding binding moieties are incompatible with certainaspects of the invention, e.g., those embodiments where effectormolecule release is based on free intracellular SDC-TRAP molecules.

In one embodiment, SDC-TRAPs have favorable safety profiles, forexample, when compared to, for example, the binding moiety or effectormolecule alone. One reason for the increased safety profile is the rapidclearance of SDC-TRAP molecules that do not enter into a target cell.

A number of exemplary SDC-TRAP molecules are set forth in the examples.Specifically a number of Hsp90-specific SDC-TRAP molecules are describedand used to demonstrate the efficacy of SDC-TRAP molecules.

Binding Moieties

A primary role of a binding moiety is to ensure that the SDC-TRAPdelivers its payload—the effector moiety—to its target by binding to amolecular target in or on a target cell or tissue. In this respect, itis not necessary that the binding moiety also have an effect on thetarget (e.g., in the case of an Hsp90-targeting moiety, to inhibit Hsp90in the manner that Hsp90 is are known to do, that is, exhibitpharmacological activity or interfere with its function), but in someembodiments, the binding moiety does have an effect on the target.Accordingly, in various embodiments, an activity of the SDC-TRAP is duesolely to the effector moiety exerting a pharmacological effect on thetarget cell(s), which has been better facilitated by the pharmaceuticalconjugate targeting the target cell(s). In other embodiments, anactivity of the SDC-TRAP is due in part to the binding moiety—that is,the binding moiety can have an effect beyond targeting.

The molecular target of a binding moiety may or may not be part of acomplex or structure of a plurality of biological molecules, e.g.,lipids, where the complexes or structures may include lipoproteins,lipid bilayers, and the like. However, in many embodiments, themolecular target to which the binding moiety binds will be free (e.g.,cytoplasmic globular protein and/or not be part of a macromolecularassembly or aggregation). The present invention can exploit theselectively high presence of a molecular target in locations of highphysiological activity (e.g., Hsp90 in oncological processes). Forexample, where a drug target is an intracellular drug target, acorresponding molecular target (e.g., Hsp90) can be present in the cell.Likewise, where a drug target is an extracellular drug target, acorresponding molecular target (e.g., Hsp90) can be extracellular,proximal, or associated with the extracellular cell membrane of thetarget cell or tissue.

In various embodiments, a binding moiety can effect a target cell ortissue (e.g., in the case of an Hsp90-targeting moiety that in factinhibits Hsp90, for example, Hsp90i). In such embodiments, apharmacological activity of the binding moiety contributes to,complements, or augments, the pharmacological activity of the effectormoiety. Such embodiments go beyond the advantages combination therapies(e.g., a cancer combination therapy of Hsp90i and a second drug such asganetespib or crizotinib) by providing a therapy that can be carried outby administration of a single SDC-TRAP that realizes both the benefitsof the combination therapy and targeting. Other examples of suchSDC-TRAPs include conjugates of an Hsp90i (such as ganetespib) and asecond cancer drug such as LY2801653, LY2835219, LY2606368, LY2874455,LY2090314, LY2940680, LY278544, LY2228820, LY2157299, LY2603618,LY353381, and trimetrexate.

A range of pharmaceutical activities can be achieved by judiciousselection of a binding moiety and an effector moiety. For example, fortreating solid tumors, e.g., colon cancer, high continuous doses ofantimetabolites such as capecitabine or gemcitabine tend to be requiredin combination with other drugs. A conjugate having an Hsp90-targetingmoiety with lower binding affinity or inhibitory activity to Hsp90,e.g., as determined by a HER2 degradation assay, can be designed to meetthis need. Such a conjugate can comprise an effector moiety that is astrong, potent antimetabolite such as 5-FU, to afford a high dose of theconjugate that may be dosed relatively frequently. Such an approach notonly achieves the aim of providing a high dose of an antimetabolitefragment at the tumor, but also lowers the toxicity of administering thedrug on its own, owing to the plasma stability of SDC-TRAPs of theinvention, and the ability of the Hsp90-targeting moiety to deliver theantimetabolite to the desired cells or tissues.

In embodiments where solid tumors such as SCLC or colorectal cancer areto be treated with drugs such as topotecan or irinotecan, only low dosesof the drug may be dosed. Due to the very high intrinsic activity ofthese drugs, an SDC-TRAP should be designed to provide a low dose ofsuch drugs at the target tissue. In this scenario, for example, anHsp90-targeting moiety having a higher binding affinity or inhibitoryactivity to Hsp90 (e.g., as determined by a HER2 degradation assay) cansufficiently maintain the presence of the drug in the tissue at a veryhigh level, to ensure that enough of the drug reaches and is retained bythe desired target tissue due to the low dosing.

In various illustrative embodiments where a molecular target of abinding moiety is Hsp90, the binding moiety can be an Hsp90-targetingmoiety, for example a triazole/resorcinol-based compound that bindsHsp90, or a resorcinol amide-based compound that binds Hsp90, e.g.,ganetespib, AUY-922 or AT-13387. In another embodiment, the bindingmoiety may advantageously be an Hsp90-binding compound of formula (I):

wherein

R¹ may be alkyl, aryl, halide, carboxamide or sulfonamide; R² may bealkyl, cycloalkyl, aryl or heteroaryl, wherein when R² is a 6 memberedaryl or heteroaryl, R² is substituted at the 3- and 4-positions relativeto the connection point on the triazole ring, through which a linker Lis attached; and R³ may be SH, OH, —CONHR⁴, aryl or heteroaryl, whereinwhen R³ is a 6 membered aryl or heteroaryl, R³ is substituted at the 3or 4 position.

In another embodiment, the binding moiety may advantageously be anHsp90-binding compound of formula (II):

wherein

R¹ may be alkyl, aryl, halo, carboxamido, sulfonamido; and R² may beoptionally substituted alkyl, cycloalkyl, aryl or heteroaryl. Examplesof such compounds include5-(2,4-dihydroxy-5-isopropylphenyl)-N-(2-morpholinoethyl)-4-(4-(morpholinomethyl)phenyl)-4H-1,2,4-triazole-3-carboxamideand5-(2,4-dihydroxy-5-isopropylphenyl)-4-(4-(4-methylpiperazin-1-yl)phenyl)-N-(2,2,2-trifluoroethyl)-4H-1,2,4-triazole-3-carboxamide.

In another embodiment, the binding moiety may advantageously be anHsp90-binding compound of formula (III):

wherein

X, Y, and Z may independently be CH, N, O or S (with appropriatesubstitutions and satisfying the valency of the corresponding atoms andaromaticity of the ring); R¹ may be alkyl, aryl, halide, carboxamido orsulfonamido; R² may be substituted alkyl, cycloalkyl, aryl orheteroaryl, where a linker L is connected directly or to the extendedsubstitutions on these rings; R³ may be SH, OH, NR⁴R⁵ AND —CONHR⁶, towhich an effector moiety may be connected; R⁴ and R⁵ may independentlybe H, alkyl, aryl, or heteroaryl; and R⁶ may be alkyl, aryl, orheteroaryl, having a minimum of one functional group to which aneffector moiety may be connected. Examples of such compounds includeAUY-922:

In another embodiment, the binding moiety may advantageously be anHsp90-binding compound of formula (IV):

wherein

R¹ may be alkyl, aryl, halo, carboxamido or sulfonamido; R² and R³ areindependently C₁-C₅ hydrocarbyl groups optionally substituted with oneor more of hydroxy, halogen, C₁-C₂ alkoxy, amino, mono- and di-C₁-C₂alkylamino; 5- to 12-membered aryl or heteroaryl groups; or, R² and R³,taken together with the nitrogen atom to which they are attached, form a4- to 8-membered monocyclic heterocyclic group, of which up to 5 ringmembers are selected from O, N and S. Examples of such compounds includeAT-13387:

In certain embodiments, to enhance the bioavailability or delivery ofthe pharmaceutical conjugate, the binding moiety may be a prodrug of theHsp90-binding compound. FIG. 1 shows how the illustrated Hsp90-targetingmoiety may be suitably modified at one or more positions to enhance thephysical, pharmacokinetic or pharmacodynamic properties of theconjugate.

Specific examples of suitable Hsp90-targeting moieties includegeldanamycins, e.g., IPI-493

macbecins, tripterins, tanespimycins, e.g., 17-AAG

KF-55823

radicicols, KF-58333

KF-58332

17-DMAG

IPI-504

BIIB-021

BIIB-028, PU-H64

PU-H71

PU-DZ8

PU-HZ151

SNX-2112

SNX-2321

SNX-5422

SNX-7081

SNX-8891, SNX-0723

SAR-567530, ABI-287, ABI-328, AT-13387

NSC-113497

PF-3823863

PF-4470296

EC-102, EC-154, ARQ-250-RP, BC-274

VER-50589

KW-2478

BHI-001, AUY-922

EMD-614684

EMD-683671, XL-888, VER-51047

KOS-2484, KOS-2539, CUDC-305

MPC-3100

CH-5164840

PU-DZ13

PU-HZ151

PU-DZ13

VER-82576

VER-82160

VER-82576

VER-82160

NXD-30001

NVP-HSP990

SST-0201CL1

SST-0115AA1

SST-0221AA1

SST-0223AA1

novobiocin (a C-terminal Hsp90i.) The selection of other Hsp90-targetingmoieties will be within the grasp of one of ordinary skill in the art.Likewise, the selection of binding moieties suitable for other moleculartargets and/or other applications will be within the ability of one ofordinary skill in the art.

Additionally Hsp90 targeting moieties can be used to construct SDC-TRAPmolecules for the treatment of inflammation. For example, bindingmoieties comprising the compounds shown in Tables 5, 6, and 7 of U.S.Patent Publication 2010/0280032, which is incorporated herein byreference in its entirety, or compounds of any formula therein, ortautomers, pharmaceutically acceptable salts, solvates, clathrates,hydrates, polymorphs or prodrugs thereof, inhibit the activity of Hsp90and, thereby cause the degradation of Hsp90 client proteins. Any ofthese compounds may be coupled to an effector molecule to form anSDC-TRAP. The glucocorticoid receptor is a client protein of Hsp90 andbinds to Hsp90 when it is in the conformation that is able to bindglucocorticoid ligands such as cortisol. Once a glucocorticoid binds toGR, the receptor disassociates with Hsp90 and translocates to thenucleus where it modulates gene expression to reduce inflammatoryresponses such as proinflammatory cytokine production. Thus,glucocorticoids may be given to patients in need of immunosuppressionand patients with inflammatory and autoimmune disorders. Unfortunately,although glucocorticoids are effective at relieving inflammation, theyhave a number of severe side effects including osteoporosis, musclewasting, hypertension, insulin resistance, truncal obesity and fatredistribution, and inhibition of wound repair Inhibition of Hsp90causes changes in GR activity which results in reduction of inflammatoryresponses similar to those seen for glucocorticoids. However, since themechanism for reducing inflammation is different than that ofglucocorticoids, it is expected that some or all of the side effects ofglucocorticoid treatment will be reduced or eliminated.

Effector Moieties

An effector moiety can be any therapeutic or imaging agent that can beconjugated to a binding moiety and, in a thus conjugated state,delivered to a molecular target of the binding moiety. An effectormolecule can, in some cases, require a linking moiety for conjugation(e.g., cannot be directly conjugated to a binding moiety). Similarly, aneffector molecule can, in some cases, impede or reduce the ability ofthe binding moiety and/or SDC-TRAP to reach a target as long as theSDC-TRAP can still effect the target. However, in preferred embodiments,an effector moiety is readily conjugatable and may benefits delivery to,and effecting, of the target.

As described in greater detail below, an effector moiety can comprise aregion that can be modified and/or participate in covalent linkage to abinding moiety without substantially adversely affecting the bindingmoiety's ability to bind to its target. An effector moiety can be apharmaceutical molecule or a derivative thereof, which essentiallyretains activity while conjugated to a binding moiety. It will beappreciated that drugs with otherwise good and desirable activity canprove challenging to administer conventionally (e.g., due to poorbioavailability or undesirable side-effects in vivo prior to reachingtheir target)—such drugs can be “reclaimed” for use as effector moietiesin the SDC-TRAPs of the present invention.

Examples of effector moieties include: LY2801653, LY2835219, LY2606368,LY2874455, LY2090314, LY2940680, LY278544, LY2228820, LY2157299,LY2603618, LY353381, and trimetrexate. Further details regarding thesecompounds and their activities are provided below.

LY2801653

LY2801653 (C₃₀H₂₂F₂N₆O₃;N-[3-Fluoro-4-[1-methyl-6-(1H-pyrazol-4-yl)-1H-indazol-5-yloxy]phenyl]-1-(4-fluorophenyl)-6-methyl-2-oxo-1,2-dihydropyridine-3-carboxamide,MW 552.5309) is an orally bioavailable multi-kinase inhibitor withpotent activity against MET, MST1R, and other oncoproteins. LY2801653has the structure:

LY2801653 displays anti-tumor activities in mouse xenograft models.LY2801653 is currently being studied in human clinical trials (e.g.,NCT01285037) for the treatment of cancer. The HGF/MET signaling pathwayregulates a wide variety of normal cellular functions that can besubverted to support neoplasia, including cell proliferation, survival,apoptosis, scattering and motility, invasion, and angiogenesis. METover-expression (with or without gene amplification), aberrant autocrineor paracrine ligand production, and missense MET mutations aremechanisms that lead to activation of the MET pathway in tumors and areassociated with poor prognostic outcome. LY2801653 is a type-II ATPcompetitive, slow-off inhibitor of MET tyrosine kinase with adissociation constant (K_(i)) of 2 nM, a pharmacodynamic residence time(K_(off)) of 0.00132 min⁻¹ and t_(1/2), of 525 min LY2801653demonstrated in vitro effects on MET pathway-dependent cell scatteringand cell proliferation; in vivo anti-tumor effects in MET amplified(MKN45), MET autocrine (U-87MG, and KP4) and MET over-expressed (H441)xenograft models; and in vivo vessel normalization effects. LY2801653also maintained potency against 13 MET variants, each bearing asingle-point mutation. In subsequent nonclinical characterization,LY2801653 was found to have potent activity against several otherreceptor tyrosine oncokinases including MST1R, FLT3, AXL, MERTK, TEK,ROS1, DDR1/2 and against the serine/threonine kinases MKNK1/2. Thesestudies are further discussed in Yan et al., 2013 (Invest New Drugs.2013, 31:833-44. doi: 10.1007/s10637-012-9912-9. Epub 2012 Dec. 29).Assays provided therein and known in the art can be used to assess theactivity of LY2801653 alone or in a SCD-TRAP conjugate provided herein.

LY2835219

LY2835219 (C₂₇H₃₂F₂N₈.CH₄O₃S; 2-Pyrimidinamine,N-[5-[(4-ethyl-1-piperazinyl)methyl]-2-pyridinyl]-5-fluoro-4-[4-fluoro-2-methyl-1-(1-methylethyl)-1H-benzimidazol-6-yl]-,methanesulfonate (1:1); MW 602.7) is an orally available cell cycle andcyclin-dependent kinase (CDK) inhibitor that potently and selectivelyinhibits of CDK4 and CDK6 with IC₅₀ of 2 nM and 10 nM, respectively.LY2835219 has the structure:

LY2835219 specifically inhibits CDK4 and 6, thereby inhibitingretinoblastoma (Rb) protein phosphorylation in early G1 Inhibition of Rbphosphorylation prevents CDK-mediated G1-S phase transition, therebyarresting the cell cycle in the G1 phase, suppressing DNA synthesis andinhibiting cancer cell growth. Overexpression of the serine/threoninekinases CDK4/6, as seen in certain types of cancer, causes cell cyclederegulation. In vivo LY2835219 saturates BBB efflux with an unboundplasma IC₅₀ of about 95 nM. The percent of dose in brain forLY2835219-MsOH is 0.5-3.9%. In both a subcutaneous and intracranialhuman glioblastoma model (U87MG), LY2835219-MsOH suppressed tumor growthin a dose-dependent manner both as a single agent, and in combinationwith temozolomide (Sanchez-Martinez et al., Molecular CancerTherapeutics: November 2011; 10:Suppl. 1doi:10.1158/1535-7163.TARG-11-B234). Assays provided therein and known inthe art can be used to assess the activity of LY2835219 alone and in anSDC-TRAP conjugate.

LY2606368

LY2606368 (C₁₈H₁₉N₇O₂;5-((5-(2-(3-aminopropoxy)-6-methoxyphenyl)-1H-pyrazol-3-yl)amino)pyrazine-2-carbonitrile,MW 365.39) is an inhibitor of checkpoint kinase 1 (chk1) withantineoplastic activity. LY2606368 is being analyzed in clinical trialsfor the treatment of advanced cancer (NCT01115790). LY2606368 has thestructure:

ChK1 is a key kinase in the DNA damage response signaling network,including cell cycle checkpoints, DNA repair, apoptosis andtranscription, thus emerging as an attractive target in anti-cancertherapy. LY2606368, a potent and selective ATP competitive inhibitor ofthe Chk1 protein kinase. LY2606368 has been reported to inhibit Chk1auto-phosphorylation activated by DNA damaging agents, and inducephosphorylation of H2AX, a DNA damage maker in multiple cancer celllines in vitro. In addition, LY2606368 has demonstrated potent singleagent activity and potentiates the anti-tumor activity of DNA damagingagents in vivo. Pancreatic cancer is one of the least curable cancers,with an approximate 5% overall 5-year survival for all patients.LY2606368 alone has been shown to significantly inhibit the cellproliferation in a variety of pancreatic cell lines (SW1990, SU86.86,Bx-PC3, AsPC-1, CFPAC-1, Capan-2, HPAF-II) with SW1990 being the mostsensitive (IC₅₀=1.5 nM). In SW1990 subcutaneous xenograft model,LY2606368 demonstrated to substantial dose-dependent inhibition of tumorgrowth. In an SW 1990 pancreas orthotopic model, which represents thelocal and metastatic growth pattern seen in pancreas cancer patients,LY2606368 treatment was demonstrated to result in over 92% inhibition ofprimary tumor growth as well as 100% inhibition of metastasis to lymphnode, spleen, and intestine. The anti-tumor effect of LY2606368treatment was further demonstrated in comparison with gemcitabine (thestandard of care for pancreatic cancer patients) in SW1990 orthotopicmodel. The anti-tumor and/or anti-metastasis mechanism of LY2606368 wasalso investigated, including phosphorylation of H2AX, cellproliferation, cell survival, and soft agar anchorage-independent growth(Wu et al, Antitumor activity of Chk1 inhibitor LY2606368 as a singleagent in SW1990 human pancreas orthotopic tumor model. Cancer Research:Apr. 15, 2012; Volume 72, Issue 8, Supplement 1 doi:10.1158/1538-7445.AM2012-1776). Assays provided therein and known in theart can be used to assess the activity of LY2606368 alone and in anSDC-TRAP conjugate.

LY2874455

LY2874455 (C₂₁H₁₉Cl₂N₅O₂;(R)-(E)-2-(4-(2-(5-(1-(3,5-Dichloropyridin-4-yl)ethoxy)-1H-indazol-3-yl)vinyl)-1H-pyrazol-1-yl)ethanol, MW 444.32) is an orally bioavailable FGF/FGFR inhibitor and iscurrently being studied in human clinical trials (e.g., NCT01212107) forthe treatment of cancer. LY2874455 has the structure:

LY2874455 is active against all 4 FGFRs, with a similar potency inbiochemical assays. It exhibits a potent activity againstFGF/FGFR-mediated signaling in several cancer cell lines and shows anexcellent broad spectrum of antitumor activity in several tumorxenograft models representing the major FGF/FGFR relevant tumorhistologies including lung, gastric, and bladder cancers and multiplemyeloma, and with a well-defined pharmacokinetic/pharmacodynamicrelationship. LY2874455 also exhibits a 6- to 9-fold in vitro and invivo selectivity on inhibition of FGF-over VEGF-mediated targetsignaling in mice. Furthermore, LY2874455 did not show VEGF receptor2-mediated toxicities such as hypertension at efficacious doses. Methodsprovided in Zhao et al. 2011 (A novel, selective inhibitor of fibroblastgrowth factor receptors that shows a potent broad spectrum of antitumoractivity in several tumor xenograft models. Mol Cancer Ther. 201110:2200-10. doi: 10.1158/1535-7163.MCT-11-0306. Epub 2011 Sep. 7.) aswell as other methods known in the art can be used to assess theactivity of LY2874455 alone and in an SDC-TRAP conjugate.

LY2090314

LY2090314 (C₂₈H₂₅FN₆O₃;3-[9-Fluoro-2-(piperidin-1-ylcarbonyl)-1,2,3,4-tetrahydropyrrolo[3,2,1-jk][1,4]benzodiazepin-7-yl]-4-(imidazo[1,2-a]pyridin-3-yl)-2,5-dihydro-1H-pyrrole-2,5-dione;MW 512.5349) is an intravenously administered glycogen synthase kinase 3beta inhibitor. LY2090314 is being analyzed in clinical trials for acuteleukemia (NCT01214603) and metastatic cancer including metastaticpancreatic cancer in combination with chemotherapy (NCT01287520,NCT01632306). LY2090314 has the structure:

In vitro, LY2090314 is a small molecule, tight-binding, ATP-competitiveinhibitor of GSK3β. Methods provided, for example, in Watanabe et al.2012 (Selective growth inhibition by glycogen synthase kinase-3inhibitors in tumorigenic HeLa hybrid cells is mediated throughNF-κB-dependent GLUT3 expression. Oncogenesis. 1: e21. Published online2012 July 9. doi: 10.1038/oncsis.2012.21) as well as other methods knownin the art can be used to assess the activity of LY2090314 alone and inan SDC-TRAP conjugate.

LY2940680

LY2940680 (C₂₆H₂₄F₄N₆O—ClH;4-Fluoro-N-methyl-N-[1-[4-(1-methyl-1H-pyrazol-5-yl)phthalazin-1-yl]piperidin-4-yl]-2-(trifluoromethyl)benzamidehydrochloride; MW 548.693) is a smoothened antagonist for the treatmentof cancer with deregulated hedgehog signaling. LY2940680 is beinganalyzed in clinical trials for small cell lung cancer (NCT01722292),pediatric medulloblastoma or rhabdomyosarcoma (NCT01697514), andadvanced cancers (NCT01919398 and NCT01226485). LY2940680 has thestructure:

The Hedgehog (Hh) pathway is a highly conserved signaling system thatplays an important role in embryonic development and tissue homeostasisthrough regulation of cell differentiation and proliferation, andderegulated Hh signaling has been implicated in variety of cancers. Twodistinct mechanisms are responsible for inappropriate and uncontrolledHh pathway activation in human malignancies: ligand-dependent, due toover-expression of Hh ligand, and ligand-independent, resulting fromgenetic mutations in pathway components such as Patched (Ptch) andSmoothened (Smo). Smo, a member of the class F G-protein coupledreceptor family, is a key regulator of Hh signaling pathway, andtherefore is an attractive target for pathway modulation. LY2940680binds to the Smo receptor and potently inhibits Hh signaling in Daoy, ahuman medulloblastoma tumor cell line, and C3H10T½, a mouse mesenchymalcell line. LY2940680 binds to and inhibits the functional activity ofresistant Smo mutant (D473H) produced by treatment with GDC-0449 (a Smoantagonist from Genentech). Treatment of Ptch^(+/−) p53^(−/−) transgenicmice, which spontaneously develop medulloblastoma, with oraladministration of LY2940680 produced substantial efficacy andsignificantly improved their survival. Magnetic resonance imaging ofthese mice revealed rapid kinetics of anti-tumor activity.Immunohistochemistry analysis of medulloblastoma tumors showed thatLY2940680 treatment induced Caspase-3 activity and reducedproliferation. LY2940680 inhibited Hh regulated gene expression in thesubcutaneous xenograft tumor stroma and produced significant anti-tumoractivity (Bender et al., 2011. Abstract 2819: Identification andcharacterization of a novel smoothened antagonist for the treatment ofcancer with deregulated hedgehog signaling. Cancer Research:8:Supplement 1 doi: 10.1158/1538-7445.AM2011-2819). Assays providedtherein and known in the art can be used to assess the activity ofLY2940680 alone and in an SDC-TRAP conjugate.

LY2784544 (Gandotinib)

LY2784544 (Gandotinib; C₂₃H₂₅ClFN₇O;3-(4-Chloro-2-fluorobenzyl)-2-methyl-N-(5-methyl-1H-pyrazol-3-yl)-8-(morpholin-4-ylmethyl)imidazo[1,2-b]pyridazin-6-amine;MW 469.942) is an orally bioavailable JAK2 specific kinase inhibitor forthe treatment of myeloproliferative disorders. LY2784544 is beinganalyzed in clinical trials for the treatment of myeloproliferativedisorders including myeloproliferative neoplasms (NCT01134120;NCT01520220; NCT01594723). LY2784544 has the structure:

LY2784544 was discovered and characterized using a JAK2-inhibitionscreening assay in tandem with biochemical and cell-based assays.LY2784544 is selective for JAK2 in vitro and effectively inhibitsJAK2V617F-driven signaling and cell proliferation in Ba/F3 cells(IC₅₀=20 and 55 nM, respectively). In comparison, LY2784544 was muchless potent at inhibiting interleukin-3-stimulated wild-typeJAK2-mediated signaling and cell proliferation (IC50=1183 and 1309 nM,respectively). In vivo, LY2784544 was shown to inhibit STATSphosphorylation in Ba/F3-JAK2V617F-GFP (green fluorescent protein)ascitic tumor cells (TED₅₀=12.7 mg/kg) and was shown to significantlyreduce (P<0.05) Ba/F3-JAK2V617F-GFP tumor burden in theJAK2V617F-induced MPN model (TED₅₀=13.7 mg/kg, twice daily). Incontrast, LY2784544 was found to have no effect on erythroidprogenitors, reticulocytes or platelets (Ma et al., 2013, Discovery andcharacterization of LY2784544, a small-molecule tyrosine kinaseinhibitor of JAK2V617F. Blood Cancer Journal (2013) 3, e109;doi:10.1038/bcj.2013.6

Published online 12 Apr. 2013). Assays provided therein and known in theart can be used to assess the activity of LY2784544 alone and in anSDC-TRAP conjugate.

LY2228820 (Ralimetinib)

LY2228820 (Ralimetinib; C₂₄H₂₉FN₆-2CH₄O₃S;5-[2-tert-Butyl-4-(4-fluorophenyl)-1H-imidazol-5-yl]-3-(2,2-dimethylpropyl)-3H-imidazo[4,5-b]pyridin-2-aminedimethanesulfonate; MW 612.737) is a tri-substituted imidazolederivative that is a potent and ATP-competitive inhibitor of the α and βisoforms of p38 MAPK in vitro (IC₅₀=5.3 nM and 3.2 nM, respectively).LY2228820 is being studied in clinical trials for treatment of advancedcancer (NCT01393990) and recurrent ovarian cancer (NCT01663857).LY2228820 has the structure:

LY2228820 dimesylate is a tri-substituted imidazole derivative that is apotent and ATP-competitive inhibitor of the α and β isoforms of p38 MAPKin vitro (IC₅₀=5.3 nM and 3.2 nM, respectively). This compounddisplays >1000-fold selectivity for p38α MAPK versus 179 other kinasestested (including p38δ and γ isoforms). In cell-based assays, LY2228820dimesylate was found to potently and selectively inhibit phosphorylationof MK2 (Thr334) in TNFα-stimulated HeLa cells (IC₅₀=8.1 nM) andanisomycin-induced mouse RAW264.7 macrophages (IC₅₀=35.3 nM) with nochanges in phosphorylation of p38a MAPK, JNK, ERK1/2, c-jun, ATF2 orcMyc at concentrations up to 14M. LY2228820 dimesylate has beendemonstrated to reduce TNFα secretion by LPS/IFNγ-stimulated macrophages(IC₅₀=6.3 nM). In mice transplanted with B16-F10 melanomas, phospho-MK2was found to be effectively inhibited by LY2228820 dimesylate in tumorsin a dose-dependent manner (TMED₇₀=19.4 mg/kg). Significant targetinhibition (>40% inhibition of phospho-MK2) was maintained for approx.4-8 hrs following a single 10 mg/kg oral dose. In a broad range ofxenograft models (A-549 NSCLC, SK-OV-3 Ovarian, U-87MG Glioma,MDA-MB-468 Breast), LY2228820 dimesylate demonstrated significant tumorgrowth delay (Campbell, et al., 2011. Abstract B235: Characterization ofLY2228820 dimesylate, a potent and selective inhibitor of p38 MAPK withantitumor activity. Molecular Cancer Therapeutics 10:Suppl. 1. doi:10.1158/1535-7163.TARG-11-B235). Assays provided therein and known inthe art can be used to assess the activity of LY2228820 alone and in anSDC-TRAP conjugate.

LY2157299 (Galunisertib)

LY2157299 (Galunisertib; C₂₂H₁₉N₅O;4-(2-(6-methylpyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)quinoline-6-carboxamide;MW 369.42) is a potent TGFβ receptor I (TβRI) inhibitor with IC50 of 56nM. LY2157299 is being studied in clinical trials for treatment ofglioma and glioblastoma (NCT01220271 and NCT01582269) and metastaticcancer including metastatic pancreatic cancer (NCT01373164). LY2157299has the structure:

LY2157299 has been demonstrated to potently inhibit the TGFβ receptorsignaling and abolishes the TGFβ induced Smad2 phosphorylation in HUVECcells. LY2157299 has also shown dose dependent potentiation of VEGF orbFGF induced cell proliferation in HUVEC and promotes VEGF induced HUVECcell migration. LY2157299 has been shown to potentiate angiogenesis inthe in vitro VEGF-stimulated cord formation assay. LY2157299 has beendemonstrated to inhibit TGF-β-mediated SMAD2 activation andhematopoietic suppression in primary hematopoietic stem cells in adose-dependent manner and to stimulate hematopoiesis from primary MDSbone marrow specimens. In human glioblastoma (GBM) cells, LY2157299 hasbeen shown to block signaling through the heteromeric TGFβ receptorcomplex to reduce levels of active, phosphorylated SMAD. LY2157299 hasalso been demonstrated to inhibit HCC cell migration on Laminin-5,Fibronectin, Vitronectin, Fibrinogen and Collagen-I and de novophosphorylation of pSMAD2. LY2157299 has also been shown to inhibit HCCmigration and cell growth independently of the expression levels ofTGF-βRII (Dituri et al., 2013. Differential Inhibition of the TGF-βSignaling Pathway in HCC Cells Using the Small Molecule InhibitorLY2157299 and the D10 Monoclonal Antibody against TGF-β Receptor TypeII. PLoS One. 2013 Jun. 27; 8(6):e67109. Print 2013). Assays providedtherein and known in the art can be used to assess the activity ofLY2157299 alone and in an SDC-TRAP conjugate.

LY2603618 (Rabusertib)

LY2603618 (Rabusertib; C₁₈H₂₂BrN₅O₃;1-{5-Bromo-4-methyl-2-[(2S)-2-morpholinylmethoxy]phenyl}-3-(5-methyl-2-pyrazinyl)urea;MW 436.303, also known as Rabusertib) is an inhibitor of the cell cyclecheckpoint kinase 1 (chk1) and checkpoint kinase 2 (chk2). LY2603618 isbeing studied in clinical trials for treatment of advanced or metastatictumors (NCT01296568), non-small cell lung cancer (NCT01139775),pancreatic cancer (NCT00839332), solid tumors NCT01341457); andnon-small cell lung cancer (NCT00988858). Y2603618 has the structure:

Chk2, an ATP-dependent serine-threonine kinase, is a key component inthe DNA replication-monitoring checkpoint system and is activated bydouble-stranded breaks (DSBs); activated chk2 is overexpressed by avariety of cancer cell types. Chk2 may prevent the repair of DNA causedby DNA-damaging agents, thus potentiating the antitumor efficacies ofvarious chemotherapeutic agents. Pharmacological inhibition of Chk1 inthe absence of p53 functionality leads to abrogation of DNA damagecheckpoints and has been shown preclinically to enhance the activity ofmany standard of care chemotherapeutic agents. LY2603618 is a potent andselective small molecule inhibitor of Chk1 protein kinase activity invitro (IC50=7 nM) and the first selective Chk1 inhibitor to enterclinical cancer trials. Treatment of cells with LY2603618 produces acellular phenotype similar to that reported for depletion of Chk1 byRNAi. Inhibition of intracellular Chk1 by LY2603618 results in impairedDNA synthesis, elevated H2A.X phosphorylation indicative of DNA damageand premature entry into mitosis. When HeLa cells are exposed todoxorubicin to induce a G2/M checkpoint arrest, subsequent treatmentwith LY2603618 releases the checkpoint, resulting in cells entering intometaphase with poorly condensed chromosomes. Consistent with abrogationof the Chk1 and p53-dependent G2/M checkpoint, mutant TP53 HT-29 coloncancer cells were found to be more sensitive to gemcitabine when alsotreated with LY2603618, while wild-type TP53 HCT116 cells were notsensitized by LY2603618 to gemcitabine. Treatment of Calu-6 human mutantTP53 lung cancer cell xenografts with gemcitabine results in astimulation of Chk1 kinase activity that was inhibited byco-administration of LY2603618 (King et al., 2013. Characterization andpreclinical development of LY2603618: a selective and potent Chk1inhibitor. Investigational New Drugs). Assays provided therein and knownin the art can be used to assess the activity of LY2603618 alone and inan SDC-TRAP conjugate.

LY353381 (Arzoxifene)

LY353581 (Arzoxifene; C₂₈H₂₉NO₄S;2-(4-methoxyphenyl)-3-(4-(2-(piperidin-1-yl)ethoxy)phenoxy)benzo[b]thiophen-6-olMW 475.60) is a selective estrogen receptor modulator. LY353581 has beenstudied in clinical trials for the treatment of breast cancer(NCT00005886; NCT00003428; NCT00034125) and endrometrial cancer(NCT00003669). LY353581 has the structure:

Methods to assess the activity of estrogen receptor modulators includingLY353381 are well known in the art (see, e.g., Sato et al., 1998,LY353381.HCl: a novel raloxifene analog with improved SERM potency andefficacy in vivo. J Pharmacol Exp Ther. 287:1-7). Assays providedtherein and known in the art can be used to assess the activity ofLY353381 alone and in an SDC-TRAP conjugate.

Trimetrexate

Trimetrexate (C₁₉H₂₃N₅O₃;(5-methyl-6-4(3,4,5-trimethoxyphenyl)amino)methyl)quinazoline-2,4-diamine;MW 369.42) is a quinazoline derivative and a dihydrofolate reductaseinhibitor. Trimetrexate has been studied in a number of human clinicaltrials for conditions including pneumocystis carinii pneumonia(NCT00001016), osteosarcoma (NCT00119301), colorectal cancer,(NCT00003446), pediatric cancers (NCT00002738), and pancreatic cancer(NCT00002955). Trimetrexate has the structure:

Methods to assess the activity of folate antagonists are well known inthe art (see, e.g., Fry and Jackson, 1987, Biological and biochemicalproperties of new anticancer folate antagonists. Cancer Metastasis Rev.5:251-70). Assays provided therein and known in the art can be used toassess the activity of trimetrexate alone and in an SDC-TRAP conjugate.

The effector moiety may be obtained from a library of naturallyoccurring or synthetic molecules, including a library of compoundsproduced through combinatorial means, i.e., a compound diversitycombinatorial library. When obtained from such libraries, the effectormoiety employed will have demonstrated some desirable activity in anappropriate screening assay for the activity. It is contemplated that inother embodiments, the pharmaceutical conjugate may include more thanone effector moiety(ies), providing the medicinal chemist with moreflexibility. The number of effector moieties linked to the bindingmoiety (e.g., Hsp90-targeting moiety) will generally only be limited bythe number of sites on the binding moiety (e.g., Hsp90-targeting moiety)and/or any linking moiety available for linking to an effector moiety;the steric considerations, e.g., the number of effector moieties thancan actually be linked to the binding moiety (e.g., Hsp90-targetingmoiety); and that the ability of the pharmaceutical conjugate to bind tothe molecular target (e.g., Hsp90 protein) is preserved. An example of atwo-effector moiety pharmaceutical conjugate can be seen in FIG. 2.

Conjugation and Linking Moieties

Binding moieties and effector moieties of the present invention can beconjugated, for example, through a linker or linking moiety L, where Lmay be either a bond or a linking group. For example, in variousembodiments, a binding moiety and an effector moiety are bound directlyor are parts of a single molecule. Alternatively, a linking moiety canprovide a covalent attachment between a binding moiety and effectormoiety. A linking moiety, as with a direct bond, can achieve a desiredstructural relationship between a binding moiety and effector moiety andor an SDC-TRAP and its molecular target. A linking moiety can be inert,for example, with respect to the targeting of a binding moiety andbiological activity of an effector moiety.

Appropriate linking moieties can be identified using the affinity,specificity, and/or selectivity assays described herein. Linkingmoieties can be selected based on size, for example, to provide anSDC-TRAP with size characteristics as described above. In variousembodiments, a linking moiety can be selected, or derived from, knownchemical linkers. Linking moieties can comprise a spacer groupterminated at either end with a reactive functionality capable ofcovalently bonding to the drug or ligand moieties. Spacer groups ofinterest include aliphatic and unsaturated hydrocarbon chains, spacerscontaining heteroatoms such as oxygen (ethers such as polyethyleneglycol) or nitrogen (polyamines), peptides, carbohydrates, cyclic oracyclic systems that may possibly contain heteroatoms. Spacer groups mayalso be comprised of ligands that bind to metals such that the presenceof a metal ion coordinates two or more ligands to form a complex.Specific spacer elements include: 1,4-diaminohexane, xylylenediamine,terephthalic acid, 3,6-dioxaoctanedioic acid,ethylenediamine-N,N-diacetic acid,1,1′-ethylenebis(5-oxo-3-pyrrolidinecarboxylic acid),4,4′-ethylenedipiperidine. Potential reactive functionalities includenucleophilic functional groups (amines, alcohols, thiols, hydrazides),electrophilic functional groups (aldehydes, esters, vinyl ketones,epoxides, isocyanates, maleimides), functional groups capable ofcycloaddition reactions, forming disulfide bonds, or binding to metals.Specific examples include primary and secondary amines, hydroxamicacids, N-hydroxysuccinimidyl esters, N-hydroxysuccinimidyl carbonates,oxycarbonylimidazoles, nitrophenylesters, trifluoroethyl esters,glycidyl ethers, vinylsulfones, and maleimides. Specific linkingmoieties that may find use in the SDC-TRAPs include disulfides andstable thioether moieties.

In some embodiments, the linker or linking moiety of an SDC-TRAP can beadvantageous when compared to the limited linking chemistry ofantibody-drug conjugates (ADC). For example, unlike ADCs that arelimited by the need to maintain the structure and/or stability of anantibody, SDC-TRAPs can use a wider range of linking chemistries and/orsolvents (e.g., that can alter, distort, or denature an antibody).

In various embodiments, a linking moiety is cleavable, for exampleenzymatically cleavable. A cleavable linker can be used to release aneffector moiety inside a target cell after the SDC-TRAP is internalized.The susceptibility of a linking moiety to cleavage can be used tocontrol delivery of an effector molecule. For example, a linking moietycan be selected to provide extended or prolonged release of an effectormoiety in a target cell over time (e.g., a carbamate linking moiety maybe subject to enzymatic cleavage by a carboxylesterase via the samecellular process used to cleave other carbamate prodrugs likecapecitabine or irinotecan). In these, and various other embodiments, alinking moiety can exhibit sufficient stability to ensure good targetspecificity and low systemic toxicity, but not so much stability that itresults in lowering the potency and efficacy of the SDC-TRAP.

Exemplary linkers are described in U.S. Pat. No. 6,214,345(Bristol-Myers Squibb), U.S. Pat. Appl. 2003/0096743 and U.S. Pat. Appl.2003/0130189 (both to Seattle Genetics), de Groot et al., J. Med. Chem.42, 5277 (1999); de Groot et al. J. Org. Chem. 43, 3093 (2000); de Grootet al., J. Med. Chem. 66, 8815, (2001); WO 02/083180 (Syntarga); Carl etal., J. Med. Chem. Lett. 24, 479, (1981); Dubowchik et al., Bioorg &Med. Chem. Lett. 8, 3347 (1998) and Doronina et al. BioConjug Chem.2006; Doronina et al. Nat Biotech 2003.

Identification and Selection of Targets and Corresponding SDC-TRAPs

The present invention provides for a broad class of pharmacologicalcompounds including an effector moiety conjugated to an binding moietydirecting the effector moiety to a biological target of interest. Whiletreating cancer using an Hsp90 inhibitor binding moiety conjugated to acytotoxic agent effector moiety is one illustrative example of thepresent invention, SDC-TRAPs are fundamentally broader in terms of theircompositions and uses.

In various embodiments, the broad class of SDC-TRAP pharmacologicalcompounds that are directed to biological targets have the followingproperties:

the biological target (a cell and/or tissue target of interest, e.g., atumor) should be effectible by an effector moiety, and the effectormoiety should be known or developed for the biological target (e.g.,chemotherapeutic agent for the tumor); the biological target should beassociated with a molecular target (e.g., biomolecule, capable of beingspecifically bound, that is uniquely represented in the biologicaltarget) that specifically interacts with a binding moiety, and thebinding moiety should be known or developed for the molecular target(e.g., ligand for the biomolecule); and the effector moiety and bindingmoiety should be amenable to coupling and should essentially retaintheir respective activity after coupling. Furthermore, the conjugateshould be capable of reaching and interacting with the molecular target,and in clinical applications should be suitable for administration to asubject (e.g., a subject can tolerate a therapeutically effective dose).Examples of therapeutic molecular targets (i.e., binding moiety bindingpartners) for various conditions/disease states include LY2801653,LY2835219, LY2606368, LY2874455, LY2090314, LY2940680, LY278544,LY2228820, LY2157299, LY2603618, LY353381, and trimetrexate.

Imaging Moieties, and Diagnostic and Research Applications

In various embodiments, the effector moiety is an imaging moiety—thatis, a molecule, compound, or fragment thereof that facilitates atechnique and/or process used to create images or take measurements of acell, tissue, and/or organism (or parts or functions thereof) forclinical and/or research purposes. An imaging moiety can produce, forexample, a signal through emission and/or interaction withelectromagnetic, nuclear, and/or mechanical (e.g., acoustic as inultrasound) energy. An imaging moiety can be used, for example, invarious radiology, nuclear medicine, endoscopy, thermography,photography, spectroscopy, and microscopy methods.

Imaging studies can be used, for example, in a clinical or researchsetting to diagnose a subject, select a subject for therapy, select asubject for participation in a clinical trial, monitor the progressionof a disease, monitor the effect of therapy, to determine if a subjectshould discontinue or continue therapy, to determine if a subject hasreached a clinical end point, and to determine recurrence of a diseaseImaging studies can be used, for example, to conduct research toidentify effective interacting moieties and/or effector moieties and/orcombinations thereof, to identify effective dosing and dose scheduling,to identify effective routes of administration, and to identify suitabletargets (e.g., diseases susceptible to particular treatment).

Methods of Making Pharmaceutical Conjugates

The pharmaceutical conjugates, i.e., SDC-TRAPs, of the invention may beprepared using any convenient methodology. In a rational approach, thepharmaceutical conjugates are constructed from their individualcomponents, binding moiety, in some cases a linker, and effector moiety.The components can be covalently bonded to one another throughfunctional groups, as is known in the art, where such functional groupsmay be present on the components or introduced onto the components usingone or more steps, e.g., oxidation reactions, reduction reactions,cleavage reactions and the like. Functional groups that may be used incovalently bonding the components together to produce the pharmaceuticalconjugate include: hydroxy, sulfhydryl, amino, and the like. Theparticular portion of the different components that are modified toprovide for covalent linkage will be chosen so as not to substantiallyadversely interfere with that components desired binding activity, e.g.,for the effector moiety, a region that does not affect the targetbinding activity will be modified, such that a sufficient amount of thedesired drug activity is preserved. Where necessary and/or desired,certain moieties on the components may be protected using blockinggroups, as is known in the art, see, e.g., Green & Wuts, ProtectiveGroups in Organic Synthesis (John Wiley & Sons) (1991).

Alternatively, the pharmaceutical conjugate can be produced using knowncombinatorial methods to produce large libraries of potentialpharmaceutical conjugates which may then be screened for identificationof a bifunctional, molecule with the pharmacokinetic profile.Alternatively, the pharmaceutical conjugate may be produced usingmedicinal chemistry and known structure-activity relationships for thetargeting moiety and the drug. In particular, this approach will provideinsight as to where to join the two moieties to the linker.

A number of exemplary methods for preparing SDC-TRAP molecules are setforth in the examples. As one of skill in the art will understand, theexemplary methods set forth in the examples can be modified to makeother SDC-TRAP molecules. Additional exemplary methods for preparationand testing of SDC-TRAPs are provided in U.S. application Ser. No.13/843,771 which is incorporated herein by reference in its entirety.

Methods of Use, Pharmaceutical Preparations, and Kits

The pharmaceutical conjugates find use in treatment of a host condition,e.g., a disease condition. In these methods, an effective amount of thepharmaceutical conjugate is administered to the host, where “effectiveamount” means a dosage sufficient to produce the desired result, e.g.,an improvement in a disease condition or the symptoms associatedtherewith. In many embodiments, the amount of drug in the form of thepharmaceutical conjugate that need be administered to the host in orderto be an effective amount will vary from that which must be administeredin free drug form. The difference in amounts may vary, and in manyembodiments may range from two-fold to ten-fold. In certain embodiments,e.g., where the resultant modulated pharmacokinetic property orproperties result(s) in enhanced activity as compared to the free drugcontrol, the amount of drug that is an effective amount is less than theamount of corresponding free drug that needs to be administered, wherethe amount may be two-fold, usually about four-fold and more usuallyabout ten-fold less than the amount of free drug that is administered.

The pharmaceutical conjugate may be administered to the host using anyconvenient means capable of producing the desired result. Thus, thepharmaceutical conjugate can be incorporated into a variety offormulations for therapeutic administration. More particularly, thepharmaceutical conjugate of the present invention can be formulated intopharmaceutical compositions by combination with appropriate,pharmaceutically acceptable carriers or diluents, and may be formulatedinto preparations in solid, semi-solid, liquid or gaseous forms, such astablets, capsules, powders, granules, ointments, solutions,suppositories, injections, inhalants and aerosols. As such,administration of the pharmaceutical conjugate can be achieved invarious ways, including oral, buccal, rectal, parenteral,intraperitoneal, intradermal, transdermal, intracheal, etc.,administration. In pharmaceutical dosage forms, the pharmaceuticalconjugate may be administered alone or in combination with otherpharmaceutically active compounds.

The subject methods find use in the treatment of a variety of differentdisease conditions. In certain embodiments, of particular interest isthe use of the subject methods in disease conditions where an activeagent or drug having desired activity has been previously identified,but which active agent or drug does not bind to its target with desiredaffinity and/or specificity. With such active agents or drugs, thesubject methods can be used to enhance the binding affinity and/orspecificity of the agent for its target.

The specific disease conditions treatable by with the subjectbifunctional compounds are as varied as the types of drug moieties thatcan be present in the pharmaceutical conjugate. Thus, disease conditionsinclude cellular proliferative diseases, such as neoplastic diseases,autoimmune diseases, central nervous system or neurodegenerativediseases, cardiovascular diseases, hormonal abnormality diseases,infectious diseases, and the like.

By treatment is meant at least an amelioration of the symptomsassociated with the disease condition afflicting the host, whereamelioration is used in a broad sense to refer to at least a reductionin the magnitude of a parameter, e.g., symptom, associated with thepathological condition being treated, such as inflammation and painassociated therewith. As such, treatment also includes situations wherethe pathological condition, or at least symptoms associated therewith,are completely inhibited, e.g., prevented from happening, or stopped,e.g., terminated, such that the host no longer suffers from thepathological condition, or at least the symptoms that characterize thepathological condition.

Methods of use of the invention extend beyond strict treatment of adisease. For example, the invention includes uses in a clinical orresearch setting to diagnose a subject, select a subject for therapy,select a subject for participation in a clinical trial, monitor theprogression of a disease, monitor the effect of therapy, to determine ifa subject should discontinue or continue therapy, to determine if asubject has reached a clinical end point, and to determine recurrence ofa disease. The invention also includes uses in conducting research toidentify effective interacting moieties and/or effector moieties and/orcombinations thereof, to identify effective dosing and dose scheduling,to identify effective routes of administration, and to identify suitabletargets (e.g., diseases susceptible to particular treatment).

A variety of hosts are treatable according to the subject methods.Generally such hosts are “mammals” or “mammalian,” where these terms areused broadly to describe organisms which are within the class Mammalia,including the orders carnivore (e.g., dogs and cats), rodentia (e.g.,mice, guinea pigs, and rats), and primates (e.g., humans, chimpanzees,and monkeys). In many embodiments, the hosts will be humans

The invention provides kits for treating a subject in need thereofcomprising at least one SDC-TRAP and instruction for administering atherapeutically effective amount of the at least one SDC-TRAP to thesubject, thereby treating the subject. The invention also provides kitsfor imaging, diagnosing, and/or selecting a subject comprising at leastone SDC-TRAP and instruction for administering an effective amount of atleast one SDC-TRAP to the subject, thereby imaging, diagnosing, and/orselecting the subject.

Kits with unit doses of the pharmaceutical conjugate, usually in oral orinjectable doses and often in a storage stable formulation, areprovided. In such kits, in addition to the containers containing theunit doses, an informational package insert describing the use andattendant benefits of the drugs in treating pathological condition ofinterest will be included. Preferred compounds and unit doses are thosedescribed herein above.

The invention also provides methods for treatment of a disease ordisorder in which the subject to be treated is selected for treatmentbased on the presence of, or the overexpression of, a particularprotein. For example, subjects may be selected for treatment of cancerbased on the presence of greater the normal levels of Hsp90. In thiscase, subjects would be administered an SDC-TRAP that comprises abinding moiety that selectively binds to Hsp90.

The invention provides methods of treating or preventing an inflammatorydisorder in a subject, comprising administering to the subject aneffective amount of a compound represented by any one of formula (I)through (LXXII), or any embodiment thereof, or a compound shown in Table5, 6, or 7 as disclosed in U.S. Patent Publication 2010/0280032. In oneembodiment, the compound or binding moiety or SDC-TRAP may beadministered to a human to treat or prevent an inflammatory disorder. Inanother embodiment, the inflammatory disorder is selected from the groupconsisting of transplant rejection, skin graft rejection, arthritis,rheumatoid arthritis, osteoarthritis and bone diseases associated withincreased bone resorption; inflammatory bowel disease, ileitis,ulcerative colitis, Barrett's syndrome, Crohn's disease; asthma, adultrespiratory distress syndrome, chronic obstructive airway disease;corneal dystrophy, trachoma, onchocerciasis, uveitis, sympatheticophthalmitis, endophthalmitis; gingivitis, periodontitis; tuberculosis;leprosy; uremic complications, glomerulonephritis, nephrosis;sclerodermatitis, psoriasis, eczema; chronic demyelinating diseases ofthe nervous system, multiple sclerosis, AIDS-related neurodegeneration,Alzheimer's disease, infectious meningitis, encephalomyelitis,Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosisviral or autoimmune encephalitis; autoimmune disorders, immune-complexvasculitis, systemic lupus and erythematodes; systemic lupuserythematosus (SLE); cardiomyopathy, ischemic heart diseasehypercholesterolemia, atherosclerosis, preeclampsia; chronic liverfailure, brain and spinal cord trauma. In another embodiment, anSDC-TRAP, or a compound shown in Table 5, 6, or 7 as disclosed in U.S.Patent Publication 2010/0280032, is administered with an additionaltherapeutic agent. In another embodiment, the additional therapeuticagent may an anti-inflammatory agent.

In one embodiment, an SDC-TRAP that is administered to a subject butdoes not enter a target cell is rapidly cleared from the body. In thisembodiment, the SDC-TRAP that does not enter a target cell is rapidlycleared in order to reduce the toxicity due to the components of theSDC-TRAP, the degradation products of the SDC-TRAP or the SDC-TRAPmolecule. Clearance rate can be determined by measuring the plasmaconcentration of the SDC-TRAP molecule as a function of time.

Likewise, SDC-TRAP molecules that enter non-targeted cells by passivediffusion rapidly exit the non-targeted cell or tissue and are eithereliminated from the subject or proceed to enter and be retained atargeted cell or tissue. For example, an SDC-TRAP that is intended totreat tumor cells and is targeted to tumor cells that overexpress, forexample, Hsp90 will accumulate selectively in tumor cells thatoverexpress Hsp90. Accordingly, very low levels of this exemplarySDC-TRAP will be present in non-tumor tissue such as normal lung tissue,heart, kidney, and the like. In one embodiment, the safety of theSDC-TRAP molecules of the invention can be determined by their lack ofaccumulation in non-targeted tissue. Conversely, the safety of theSDC-TRAP molecules of the invention can be determined by their selectiveaccumulation in the targeted cells and/or tissue.

EXAMPLES

The following examples, which are briefly summarized and then discussedin turn below, are offered by way of illustration and not by way oflimitation.

Example 1 presents the synthesis of exemplary SDC-TRAPs.

Example 2 presents methods for assessing the targeted delivery ofexemplary SDC-TRAPs.

Example 3 presents an exemplary assay for HER2 degradation.

Example 4 presents an exemplary assay for toxicity of SDC-TRAPs.

Example 5 presents an exemplary assay for the stability of SDC-TRAPs inplasma.

Example 6 presents an exemplary assay for anti-tumor activity ofSDC-TRAPs in xenogeneic human tumor mouse model.

Examples 7 and 8 present exemplary assays for IC₅₀ value determinationsfor SDC-TRAPs.

Example 9 presents an exemplary Hsp90α binding assay.

Example 10 presents an exemplary HER2 degradation assay.

Example 11 presents an exemplary cytotoxicity assay.

Example 12 presents an exemplary plasma stability protocol.

Example 13 presents an exemplary tissue distribution study.

Example 14 presents an exemplary assay for use of SDC-TRAPs forprevention and treatment of skin cancers and actinic keratosis

Example 15 presents an exemplary assay for determining the permeabilityof SDC-TRAP molecules

Example 16 presents an exemplary assay for determination ofpharmacodynamics of an SDC-TRAP in a tumor model.

Example 17 presents SDC-TRAP-309 structure and synthesis.

Example 18 presents SDC-TRAP-310 structure and synthesis.

Example 19 presents SDC-TRAP-311 structure and synthesis.

Example 20 presents SDC-TRAP-312 structure and synthesis.

Example 21 presents SDC-TRAP-313 structure and synthesis.

Example 22 presents SDC-TRAP-314 structure and synthesis.

Example 23 presents SDC-TRAP-315 structure and synthesis.

Example 24 presents SDC-TRAP-316 structure and synthesis.

Example 25 presents SDC-TRAP-317 structure and synthesis.

Example 26 presents SDC-TRAP-318 structure and synthesis.

Example 27 presents SDC-TRAP-319 structure and synthesis.

Example 28 presents SDC-TRAP-320 structure and synthesis.

Example 29 presents SDC-TRAP-321 structure and synthesis.

Example 30 presents SDC-TRAP-322 structure and synthesis.

Example 31 presents SDC-TRAP-323 structure and synthesis.

Example 32 presents SDC-TRAP-324 structure and synthesis.

Example 33 presents SDC-TRAP-325 structure and synthesis.

Example 34 presents SDC-TRAP-326 structure and synthesis.

Example 35 presents SDC-TRAP-327 structure and synthesis.

Example 36 presents SDC-TRAP-328 structure and synthesis.

Example 37 presents SDC-TRAP-329 structure and synthesis.

Example 38 presents SDC-TRAP-330 structure and synthesis.

Example 39 presents SDC-TRAP-331 structure and synthesis.

Example 40 presents SDC-TRAP-332 structure.

Example 41 presents SDC-TRAP-333 structure.

Example 42 presents SDC-TRAP-334 structure.

Example 1 Exemplary Synthetic Methods for SDC-TRAPs

SDC-TRAPs of an exemplary embodiment may be prepared in the followingmanner:

(1) HDC of LY2801653

(2) HDC of CDK4/6 Dual Inhibitor LY2835219

(3) HDC of LY2606368

(4) HDC of FGFR 1-3 Inhibitor LY2874455

(5) HDC of GSK3beta Inhibitor LY-2090314

(6) HDC of Gandotinib

(7) HDC of Ralimetinib—the MAPK14 (MAPK p38 Alpha) Inhibitor:

(8) Galunisertib HDC

(9) Rabusertib HDC

(10) HDC of Arzoxifene

(11) HDC of Trimetrexate:

The conjugate synthesis schemes are exemplary. Other synthetic methods,linkers, and binding moieties can be linked to the above effectormolecules.

Example 2 Tissue Distribution and Metabolism of an Exemplary Hsp90Binding Moiety

The ability of Hsp90-targeting moieties to penetrate solid tumors andexhibit rapid clearance from normal tissues for reduced toxicity isillustrated in the following tissue distribution study with a compound,ganetespib, which may be used as an Hsp90 binding moiety.

Tissue Distribution of Ganetespib in Female CD-1 Nu/Nu Mice Bearing RERFHuman NSCLC Xenografts

Objectives:

To confirm the distribution of ganetespib in blood, livers, kidneys,brains, hearts, lungs and tumors after IV administration of ganetespibto female CD-1 nu/nu mice bearing RERF human NSCLC xenografts, and toexamine metabolic profiles of ganetespib in plasma, red blood cells, andabove tissues.

Study Outline:

Test Articles: ganetespib

Animals: female CD-1 nu/nu mice bearing RERF human NSCLC xenografts(N=3/group)

Route: IV

Dosage: 50 mg/kg

Dose level: 10 mL/kg

Formulation: 10% DMSO, 18% Cremophor RH40, 3.6% dextrose solution (DRD)

Bleeding time points: 5 min, 6, 24 hr

Collected tissues: blood (plasma and red blood cells (RBC)), liver,kidneys, brain, heart, lung, tumor

Method

Sample Preparation

Plasma and RBC

Protein precipitation: 50 μL of 10 times diluted plasma or RBC+150 μLACN (10 mM NH₄OAc), vortexed and centrifuged at 10000 rpm for 8 min; 150μL supernatant+150 μL water (10 mM NH₄OAc)

Other Tissues

Protein precipitation: 100 μL homogenized tissue (1:3 tissue: PBSbuffer)+100 μL ACN (10 mM NH₄OAc), vortexed and centrifuged at 10000 rpmfor 8 min

Bioanalysis

HPLC (ChemStation)

Column: Agilent Zorbax Eclipse XDB-C18, 4.6×150 mm, 5 μm

Mobile phase: A: water containing 10 mM NH₄OAc; B: 95% ACN containing 10mM NH₄OAc

Gradient: 95/5 A/B to 5/95 A/B in 10 min, total run time 15 min

Flow rate: 1 mL/min

Column temp.: 40° C.

Wavelength: 254 nm

Injection volume: 100 μL

Calibration curve range:

Plasma: 1-50 μM (linear regression; R²=0.9901); LLOQ=1 μM

RBC: 1-50 μM (linear regression; R²=0.9987); LLOQ=1 μM

Kidney: 1-100 μM (linear regression; R²=1.0000); LLOQ=1 μM

Lung: 1-100 μM (linear regression; R²=1.0000); LLOQ=1 μM

Heart: 1-100 μM (linear regression; R²=0.9998); LLOQ=1 μM

Liver: 1-100 μM (linear regression; R²=1.0000); LLOQ=1 μM

Tumor: 0.1-10 μM (linear regression; R²=1.0000); LLOQ=0.1 μM

LC-MS/MS (Q-Trap4000)

Polarity: positive (ESI)

Column: Phenomenex Synergi, 2.1×50 mm, 4 μm

Mobile phase: A: water containing 0.1% HCOOH; B: ACN containing 0.1%HCOOH

Gradient: 60/40 A/B to 5/95 A/B in 0.5 min, total run time 4 min

Flow rate: 0.5 mL/min

Column temp.: room temperature

Injection volume: 20 μL

Calibration curve range:

Plasma: 2.5-500 nM (linear regression; R²=0.9994); LLOQ=2.5 nM

RBC: 2.5-500 nM (linear regression; R²=0.9998); LLOQ=2.5 nM

Kidney: 2.5-500 nM (linear regression; R²=0.9993); LLOQ=2.5 nM

Lung: 2.5-500 nM (linear regression; R²=0.9993); LLOQ=2.5 nM

Heart: 2.5-500 nM (linear regression; R²=0.9997); LLOQ=2.5 nM

Liver: 2.5-500 nM (linear regression; R²=1.0000); LLOQ=2.5 nM

-   -   0.5-5 μM (linear regression; R²=0.9970); LLOQ=0.5 μM

Brain: 2.5-500 nM (linear regression; R²=0.9998); LLOQ=2.5 nM

-   -   0.5-5 μM (linear regression; R²=0.9992); LLOQ=0.5 μM

Results

Formulations

The dosing solution was confirmed to have 98.1% accuracy by HPLC.

Tissue Distribution

The concentrations of ganetespib in plasma, RBC and the tissues aresummarized in FIG. 1 at each time point.

The mean plasma concentration of ganetespib at 5 min after IV injectionwas 160 μM, highest among all the tissues studied. Thereafter, theplasma ganetespib concentration declined quickly and at 6 hr, it was0.12 μM. At 24 hr, it was below the lower limit of quantitation (LLOQ,<2.5 nM).

After IV injection, ganetespib was widely distributed to the normaltissues analyzed. At 5 min, the highest concentration of ganetespibamong the tissues was observed in kidney (57.8 μM), followed by liver(46.3 μM) and heart (36.2 μM). In brain, 0.53 μM of ganetespib wasdetected at 5 min, which was the lowest among the tissues. In all thenormal tissues, the concentrations of ganetespib decreased quickly.

Although the concentration of ganetespib in tumor at 5 min (2.35 M) waslower than that in plasma and most of the other tissues studied, itremained relatively constant up to 24 hr (0.85 μM at 24 hr). However,the in vitro IC₅₀ values of ganetespib are small, and the tumorconcentration of ganetespib at 24 hr was significantly higher than IC₅₀of in vitro HER2 assays (˜30 nM). Thus, the prolonged efficacy isexpected even after ganetespib was cleared from the blood stream.

The mean concentration of ganetespib in plasma was about 10 times higherthan that in RBC at 5 min time point, indicating that ganetespib tendsto stay in plasma rather than in RBCs.

Conculsion

Ganetespib appeared to persist longer in tumor than in plasma or anyother tissues studied. The results from this study suggest thatganetespib also has a higher binding affinity to Hsp90 from tumor cellsthan Hsp90 from normal cells, and that it is possible for ganetespib tomodulate relative protein concentrations of Hsp90 and its clientproteins selectively in tumors. The plasma concentrations of ganetespibdid not correlate to the concentrations in tumor.

TABLE 1 Concentration of ganetespib in tissues: Test ganetespib ArticlesStruc- ture

Species CD-1-nu/nu female mice Tumor RERF human NSCLC Route IV Dosage 50mg/kg Form- DRD ulation kid- plasma RBS tumor liver neys brain heartlung Time (μg/mL) (μg/mL) (μg/g) (μg/g) (μg/g) (μg/g) (μg/g) (μg/g) 5min 58.4  6.00 0.86 16.9 21.1 0.19 13.2  9.24 6 hr  0.04 No data 0.29 0.14  0.06 0.07  0.05  0.05 24 hr >LLOQ  0.003 0.31  0.005  0.01 0.04 0.00  0.00 kid- plasma RBC tumor liver neys brain heart lung Time (μM)(μM) (μM) (μM) (μM) (μM) (μM) (μM) 5 min 160 16.5 2.35 46.3 57.8 0.5336.2 25.4 6 hr  0.12 N/A 0.80  0.39  0.15 0.18  0.13  0.14 24 hr >LLOQ 0.007 0.85  0.01  0.02 0.12  0.00  0.005

Summary

Ganetespib was widely distributed to various tissues. The compound wasaccumulated in tumor relative to the plasma and other tissues,indicating the higher binding affinity of this compound to Hsp90 intumor than Hsp90 in other tissues. The metabolite M2, which waspreviously thought to be human-specific, was also detected in mouseliver, kidney, heart and lung, but not in plasma. M2 does not seem to beexcreted into blood stream in mice and possibly in other species aswell.

Example 3 HER2 Degradation Assay

This example illustrates how a HER2 degradation assay may be used as atest to determine and select Hsp90-targeting moieties suitable for usein SDC-TRAPs of the invention, and further illustrates the ability ofSDC-TRAPs to target cells preferentially expressing Hsp90. Such a testmay further be used to determine the Hsp90 binding ability of SDC-TRAPsof the invention, as well as through competitive binding assays andcell-based Hsp90 client protein degradation assays known in the art.

Degradation of HER2 in Cells after Treatment with an SDC-TRAP of theinvention

Method 1:

BT-474 cells are treated with 0.5 μM, 2 μM, or 5 μM of 17-AAG (apositive control) or 0.5 μM, 2 μM, or 5 μM of an Hsp90-targeting moietyor conjugate of the invention overnight in DMEM medium. After treatment,each cytoplasmic sample is prepared from 1×10⁶ cells by incubation ofcell lysis buffer (#9803, Cell Signaling Technology) on ice for 10minutes. The resulting supernatant used as the cytosol fractions isdissolved with sample buffer for SDS-PAGE and run on a SDS-PAGE gel,blotted onto a nitrocellulose membrane by using semi-dry transfer.Non-specific binding to nitrocellulose is blocked with 5% skim milk inTBS with 0.5% Tween at room temperature for 1 hour, then probed withanti-HER2/ErB2 mAb (rabbit IgG, #2242, Cell Signaling) and anti-Tubulin(T9026, Sigma) as housekeeping control protein. HRP-conjugated goatanti-rabbit IgG (H+L) and HRP-conjugated horse anti-mouse IgG (H+L) areused as secondary Ab (#7074, #7076, Cell Signaling) and LumiGLO reagent,20× Peroxide (#7003, Cell Signaling) is used for visualization. TheHsp90 client protein HER2 is degraded when cells are treated withHsp90-targeting moieties or SDC-TRAPs of the invention. 0.5 μM of17-AAG, a known Hsp90 inhibitor used as a positive control, causespartial degradation of HER2.

Method 2:

BT-474 cells are plated in the interior 60 wells of a 96 well blackclear bottom plate (20,000 cells/well) in DMEM medium, with DMEM mediain the surrounding 36 wells, and incubated at 37° C. with 5% CO₂overnight. On the second day, concentration response curve source platesare produced (10 point, 3-fold dilution of compounds in DMSO) followedby a 1:30 dilution in an intermediate dilution plate containing DMEM.Compound is transferred from the intermediate plate to the cell plate ata dilution of 1:10. The cells are then incubated at 37° C. with 5% CO₂for 24 hours.

Cells are then fixed in 4% phosphate-buffered paraformaldehyde for 30minutes at room temperature and then permeabilized by washing five timeswith 0.1% Triton X-100 in PBS for 5 minutes at room temperature on ashaker. Cells are blocked with Odyssey Blocking Buffer (LI-COR,#927-40000) on a shaker at room temperature for 1.5 hours, followed byincubation with HER2 antibody (CST, #2165) diluted 1:400 in blockingbuffer overnight on a shaker at 4° C. Cells are washed five times with0.1% Tween-20 in PBS for 5 minutes at room temperature on a shaker andincubated with fluorescently-labeled secondary antibody (LI-COR,#926-32211) diluted 1:1000 in blocking buffer, and DRAQS nuclear stain(Biostatus Limited, #DRAQS) diluted 1:10,000, at room temperature on ashaker for 1 hour. Cells are washed 5 times with 0.1% Tween-20 in PBSfor 5 minutes at room temperature on a shaker and imaged on a LI-COROdyssey imaging station. The raw data is normalized to DRAQS and theHER2 EC₅₀ is calculated using XLfit™. The above procedures are utilizedto generate HER2 degradation data, which show the ability of variousSDC-TRAPs to target cells preferentially expressing Hsp90.

Example 4 Cytotoxicity Assay

Cell Lines.

Human H3122 NSCLC cells are obtained and grown in RPMI in the presenceof fetal bovine serum (10%), 2 mM L-glutamine and antibiotics (100 IU/mlpenicillin and 100 μg/ml streptomycin, Sigma Aldrich.) Cells aremaintained at 37° C., 5% CO₂ atmosphere.

Cell Viability Assays.

Cell viability is measured using the CellTiter-Glo® assay

(Promega). In brief, cells are plated in 96-well plates in triplicate atoptimal seeding density (determined empirically) and incubated at 37°C., 5% CO₂ atmosphere for 24 hr prior to the addition of drug or vehicle(0.3% DMSO) to the culture medium. At the end of the assay,CellTiter-Glo® is added to the wells per manufacturer's recommendation,shaken for two minutes and incubated for 10 minutes at room temperature.Luminescence (0.1 sec) is measured with a Victor II microplate reader(Perkin Elmer®) and the resulting data are used to calculate cellviability, normalized to vehicle control.

Cells as described above are treated with exemplary SDC-TRAPs and theirviability is determined as above as well. These assays demonstrate thatthe SDC-TRAP effector and targeting components maintain the cytotoxicactivity against cancer cells in the conjugate.

Example 5 Assessing Stability of SDC-TRAPs in Human and Mouse Plasma

SDC-TRAPs are incubated in human and mouse plasma for 2 h at 37° C. andassayed for integrity at 0.25, 0.5, 1 and 2 h to assess the stability ofthe SDC-TRAP, e.g., as compared to the effector and targeting compounds.Compound stability is determined using any of a number of routinemethods including, for example chromatography and mass spectrometry.

Example 6 Assessing Anti-Tumor Activity of SDC-TRAPs in Xenogeneic HumanTumor Mouse Model

Xenogeneic human tumor mouse models are well known in the art in whichhuman tumors are implanted in immunocompetent mice. Various agents aretested for their efficacy in promoting tumor shrinkage. Human tumor celllines for use in such models include, but are not limited to HCT-116colon cancer model and MCF-7 breast cancer model. An exemplary study isprovided for non-specified SDC-TRAP-A and SDC-TRAP-B which includeeffector A and effector B, respectively, in combination with the samebinding domain. It is understood that variations of the experimentaldesign can be readily envisioned to analyze the efficacy of SDC-TRAPs.

A xenograft tumor model is used to evaluate the anti-tumor efficacy ofSDC-TRAP-A and SDC-TRAP-B. The tumor model is established bytransplanting equivalent numbers of human tumor cells, e.g., HCT-116cells, into mice and testing the effect of the SDC-TRAPs on tumor volumeand change in tumor volume.

HCT 116 human colorectal adenocarcinoma tumor cells are purchased fromATCC. The cells are maintained in vitro as a monolayer culture inMcCoy's 5a Medium. Fetal bovine serum is added to the medium. The finalconcentration of fetal bovine serum is 10%. Cells are cultured at 37° C.and 5% CO₂. The tumor cells are routinely sub-cultured twice weekly bytrypsin-EDTA treatment. Cells in an exponential growth phase areharvested and counted for tumor inoculation.

100 18-22 g, 5-7 week old, female BALB/cA nude mice are inoculated withthe HCT 116 cells (2.0×10⁶, 1:1 with Matrigel) subcutaneously on theback of each animal (0.1 mL/mouse). When the average tumor volumereaches about 150-250 mm³, 60 of the inoculated mice are selected basedon tumor growth and randomly grouped into 6 treatment groups (10 miceper group) according to the following table. Mice that are not put ontreatment are euthanized. Animals are sourced through ShanghaiSINO-British SIPPR/BK Lab Animal Ltd, Shanghai, China. Mice are treatedas set forth in the table below:

Treatment Groups Dosage Dosage Animal Dosage Conc. Vol. Dosing GroupsNumber Treatment (mg/kg) (mg/mL) (mL/kg) Route Schedule 1 10 Vehicle NANA 10 IV Q7D × 3 2 10 SDC-TRAP-A 200 20 10 IV Q7D × 3 3 10 SDC-TRAP-A100 10 10 IV Q7D × 3 4 10 SDC-TRAP-B 94 9.4 10 IV Q7D × 3 5 10Unconjugated 67 6.7 10 IV Q7D × 3 effector-A 6 10 Unconjugated 67 6.7 10IV Q7D × 3 effector-B 7 Unconjugated 100 10 10 IV Q7D × 3 binding

Dose Preparation & Treatment Schedule

The dosing solutions of SDC-TRAP-A, SDC-TRAP-B, uncongugated effectormoiety, and unconjugated binding moiety (e.g., ganetespib) are preparedaccording to an appropriate formulation protocol and administered usingroutine methods.

Evaluation of Anti-Tumor Activity

During the treatment period, the implanted tumors are measured bycaliper twice per week. The tumors are measured for the maximum width(X) and length (Y) and the tumor volumes (V) are calculated using theformula: V=(X²Y)/2. The differences in the tumor volume between thecontrol and treatment groups are analyzed for significance using theunpaired two-tailed Student's t-test. P<0.05 is considered to bestatistically significant. The animal body weights are also weighed andrecorded twice per week. The changes in tumor volume and body weight inthe days following compound treatment are determined. The effects of theSDC-TRAPs are compared to each other, to the effector and binding moietythat are used to generate that SDC-TRAPs, and untreated control toidentify an SDC-TRAP with improved efficacy in inhibiting tumor growthwithout adverse effects (e.g., excessive weight loss).

Example 7 Determination of IC₅₀ by Assessing the Effects of VariousSDC-TRAPs on Tumor Shrinkage

An appropriate cell line, e.g., H3122 cells, are seeded into in 96-wellplates at 7,500 cells/90 μL/well, and were incubated for 24 hours.SDC-TRAPs, plus a binding moiety alone (e.g., ganetespib) as a control,are serially diluted in dimethylsulfoxide (DMSO) (e.g., 3000 nM, 1000nM, 333.3 nM, 111.1 nM, 37.0 nM, 12.3 nM) into each of six wells of each96-well plate. To each well of a first set of duplicate plates plates,145 μL of media was added, and the cells are incubated. The wells of asecond set of duplicate plates (pulsed plates) are incubated for 1 hour,then the wells are rinsed 2× with fresh media to remove the conjugate,and 145 μL of media is then added to each washed well. IC₅₀ isdetermined visually under a microscope after 48 hours and 72 hoursdrug-exposure. Also at the 72 hour time point, 50 μL of the cell culturesupernatant is mixed with 50 μL of CellTiter-Glo® and the luminescenceis determined, from which an IC₅₀ for each conjugate was calculated.

Example 8 IC₅₀ of Continuous and Pulsed Exposure to SDC-TRAPs

IC₅₀ toxicity is determined for 72 hour continuous exposure to SDC-TRAPsrun in triplicate, and for duplicate pulse exposure (1 hour “pulse”exposure to conjugate compound, followed by 72 hour incubation inconjugate-free media) using H3211 cells, according to the protocol setforth in Example 7.

Example 9 Hsp90^(α) Binding Assay Protocol

An Hsp90^(α) fluorescence assay kit from BPS Bioscience (Cat #50294)containing Hsp90 recombinant enzyme, FITC-labeled geldanamycin, assaybuffer and a low binding 384-well plate is used to assay Hsp90^(α)binding. Dithiothreitol (DTT) (Cat #D0643) and bovine serum albumin(BSA) (Cat #A2153) were obtained from Sigma-Aldrich. Fluorescencepolarization is measured using a PHERAstar® microplate reader (BMGLABTECH GmbH, Ortertherg, Germany.)

The SDC-TRAP and control compounds are diluted to 1 mM in DMSO andloaded into a compound dilution plate to make 3-fold dilutions yieldinga total of 8 concentrations. 1 μL of compound is transferred from thedilution plate to the low binding assay plate provided in the assay kit.5 mL of Hsp90′ binding solution is prepared having a final concentrationof 7 ng/μL Hsp90^(α), 5 nM FITC-labeled geldanamycin, 2 mM DTT and 0.1mg/mL BSA. 49 μL of binding solution is added to each microplate well,incubated at room temperature for 1 hour, then read using the PHERAstar®microplate reader. The high control sample contains no compound plusHsp90^(α); the low control sample contains no compound and no Hsp90^(α).Percent inhibition is calculated using high control as 100% and lowcontrol as 0% inhibition. The IC₅₀ is calculated using GraphPad Prism® 4software.

Example 10 HER2 Degradation Assay with BT-474 Cell Line

HER2 has emerged as a key target for anticancer drugs due to itsintrinsic involvement in the phosphatidylinositol-3-kinase-Akt/proteinkinase B (PI3K-Akt) and the mitogen-activated protein kinase (MAPK)pathways, both of which suppress apoptosis and promote tumor cellsurvival, gene transcription, angiogenesis, cellular proliferation,migration, mitosis, and differentiation. The degradation of HER2 is ameasure of efficacy of anticancer therapeutics that target Hsp90.Accordingly, the SDC-TRAP molecules of the invention that comprise abinding moiety that binds Hsp90 are tested in the following HER2degradation assay.

BT-474 cells (human breast cancer cell line ATCC HTB-20) are obtainedfrom ATCC and seeded into 12-well tissue culture plates at 0.2×10⁶/1.8mL/well. The cells are incubated for more than 6 hours at 37° C. inDMEM+10% FBS, +1% P/S, +1.5 g/L sodium bicarbonate. Each test compoundis titrated in 4-fold dilutions from 5 μM to 78 nM with DMSO and 200 μLof the titration is added to each well of the cell plate. The DMSO finalconcentration is 0.2%. Cells are incubated overnight at 37° C. in 5%CO₂.

Media is decanted from the plate, cells are washed 1× in PBS. 400 μLtrypsin (EDTA) per well is added, and the cells are incubated for 2 to 3minutes. Cells are collected into FACS tubes containing 1 ml culturemedium to neutralize the trypsin and are centrifuged for 5 minutes at1200 rpm. Supernatant is decanted and the cells are resuspended in 5 μLFITC (anti HER2/nu)/200 μL staining buffer (1×PBS+1% FBS+0.05% SodiumAzide)/tube. Controls are 5 μL IgG isotype control and staining bufferonly. Tubes are incubated for 30 minutes in the dark at roomtemperature. 1 mL staining buffer is added to each tube and the tubesare centrifuged for 6 minutes at 1200 rpm. The supernatant is decantedand 300 μL staining buffer is added to each tube, which was store at 4°C. fpr FACS (cytometer) analysis. The cytometer readout is normalizedand the potency of each compound is evaluated with IC₅₀ calculated withXLfit™ software.

Example 11 Cytotoxicity Assay with Cancer Cell Lines

Cytotoxicity of SDC-TRAP molecules is determined in multiple cancer cellline such as the three exemplary cancer cell lines provided herein. 5000cells/100 μL/well of human breast cancer cell line BT-474 (ATCC #HTB-20)and human urinary bladder cancer cell line SW780 (ATCC# CRL-2169) and5000 cells/well of human urinary bladder cancer cell line RT-112 areseeded into 96-well flat-bottom tissue cultures plates and incubatedovernight at 37° C. in 5% CO₂. BT-474 and SW780 cells are cultured inDMEM+10% FBS, +1% P/S, +1.5 g/L sodium bicarbonate; RT-112 cells arecultured in EMEM+10% FBS, +1% P/S. SDC-TRAP-0178 is titrated by 10-folddilutions from 10 μM to 10 nM and added to the plate at 10 μL/well.Final concentration of DMSO in the cell plate is 0.25%. The plates areincubated for 72 hours at 37° C. in 5% CO₂. 80 μL of CellTiter-Glo isadded to each well, followed by room temperature incubation in the darkfor 15 minutes. Cell is determined by luminescence. IC₅₀ is calculatedusing XLfit™ software.

Example 12 Tissue Distribution Extraction Procedure for SDC-TRAP TumorSamples

SDC-TRAP molecules have the the ability to be specifically targeted todesired cells. For example, SDC-TRAP molecules can be targeted to tumorsand tumor cells in order to treat cancer. This example sets forth aprotocol to extract the SDC-TRAP molecules of the invention from tumorsamples.

A 150 ng/mL solution of an SDC-TRAP in methanol is prepared using aninternal spiking solution (500 μg/mL SDC-TRAP-0002 in DMSO). Using the10 mM stock solutions of the SDC-TRAP molecule and its Hsp90i bindingmoiety and effector moiety in DMSO, spiking solutions are prepared at0.025, 0.05, 0.1, 0.5, 1, 5, 10, 50, 100, 250, and 500 μM in DMSO. 5 μLof each spiking solution is added to a 96-deep well plate.

Quality control standards are prepared from 5 μL of 0.1, 1, and 10 μMcalibration standard spiking solution added in triplicate into 96-deepwell plate and adding 50 μL of matrix (plasma or homogenized tumor).

To prepare test samples, test plasma is diluted as needed using blankplasma. Tumor samples are pulverized in liquid nitrogen, weighed, andhomogenized in PBS at 5× volume to sample weight. 50 μL of unknownplasma or homogenized tumor sample is mixed with 5 μL of DMSO. Thesamples are extracted by precipitating calibration standards, QCstandards, and unknown samples with 200 μL of internal standardsolution. The samples are mixed by vortex at room temperature forapproximately 1.5 minutes, then centrifuge at 2-8° C. 150 μL ofsupernatant is collected and 25 μL of water added. Samples are mixed andanalyzed by LC-MS/MS.

Example 13 Plasma Stability Protocol for SDC-TRAP Compounds

An exemplary assay to analyze plasma stability of SDC-TRAPs is provided.150 ng/mL solution of SDC-TRAP-0002 in methanol is prepared using theinternal standard spiking solution. This solution is used to precipitateall plasma samples in the study. 200 μL is pipetted into a 96 deepwellplate over dry ice. 10 μL of 1 mM stock in DMSO is added to a 1.5 mLmicrofuge tube, then 990 μL of plasma. Samples are mixed by vortex, then50 μL of each sample is added in triplicate to a 96-well platecontaining internal standard solution. This was designated the 0 hourtime point sample. 250 μL of the remaining plasma sample is added toeach of four 96 deepwell plates—one per time point. Samples areincubated at 37° C. with gentle shaking for 0.25, 0.5, and 1 hour. Aftereach time point, one plate of each sample is removed from the shaker andplaced on wet ice for approximately 2 minutes. 50 μL plasma aliquots (intriplicate) are added to the deepwell plate containing internal standardsolution. After the last time point is extracted, the 96 deepwell plateis vortexed, then centrifuged at 2-8° C. 150 μL of supernatant iscollected and 25 μL of water was added. Samples are mixed and analyzedby LC-MS/MS.

Example 14 Identification and Use of SDC-TRAP for Prevention andTreatment of Skin Cancers and Actinic Keratosis

Skin cancers (neoplasms) are named after the type of skin cell fromwhich they arise. Skin cancers include basal cell carcinoma, squamouscell carcinoma, malignant melanomas, and Bowen's disease. Actinickeratosis can be, but is not always, a precursor to squamous cellcarcinoma.

Drugs used for the treatment of skin cancer are selected based on thetype and severity of the skin cancer. Superficial, non-melanoma skincancers can be treated with topical agents, either alone or incombination with surgery or other therapeutic interventions. Such agentsinclude, but are not limited to, retinoids, 5-fluorouracil, diclofenac,ingenol mebutate, and imiquimod. Topical delivery permits administrationof the chemotherapeutic agent directly to the site of the tumor or skinlesion. However, the delivery of active agents into the skin can bechallenging. Moreover, many topical therapeutic agents can be irritatingto the skin, resulting in scar formation, further inhibiting thedelivery of the active agent to the site.

Imiquimod3-(2-methylpropyl)-3,5,8-triazatricyclo[7.4.0.02,6]trideca-1(9),2(6),4,7,10,12-hexaen-7-amine)is a patient-applied cream used to treat certain diseases of the skin,including skin cancers (basal cell carcinoma, Bowen's disease,superficial squamous cell carcinoma, some superficial malignantmelanomas, and actinic keratosis) as well as genital warts (condylomataacuminata). Imiquimod and its analogs activate the immune system byactivating immune cells through the toll-like receptor 7 (TLR7),commonly involved in pathogen recognition. Imiquimod can be used incombination with one or more drugs used for the treatment of skindiseases to make an SDC-TRAP molecule.

An imiquimod SDC-TRAP molecule can be formed, for example, using anyknown linker, such as those provided herein, with the desired effectormolecule. The specific linker and conjugation method used will depend,for example, on the chemical nature of the effector molecule.

Assays to determine the cytotoxicity of the imiquimod SDC-TRAP moleculesare performed using methods similar to those provided in Example 4. Cellviability assays are performed on non-transformed cells, preferably skincells, to identify SDC-TRAPs with acceptable toxicities, preferablycompounds with toxicity that is not greater than either of the parentcompounds. Cytotoxicity and skin irritation assays are also performed,for example, on pig skin, which is frequently used as a model for humanskin in toxicity/irritation assays, using routine methods.

Imiquimod SDC-TRAP molecules are also tested to confirm that theirefficacy is not inhibited by the formation of the conjugate. A number ofskin cancer cell lines are well known in the art. Dose response curvesare generated to demonstrate the efficacy of imiquimod SDC-TRAPmolecules in killing cancer cells. Preferably, the imiquimod SDC-TRAPmolecules are more effective at killing skin cancer cells than imiquimodor the effector molecule alone.

Methods to assess pharmacokinetic and pharmacodynamic properties of anagent are well known in the art. As noted above, pig skin is frequentlyused as a model for human skin, both in toxicity/irritation assays, butalso in assaying uptake and delivery of agents into skin layers andcells. Topical formulations of imiquimod, the effector molecule, andimiquimod SDC-TRAP molecules are assayed for uptake, transport throughthe skin, and persistence in the skin using routine methods.

Having identified a imiquimod SDC-TRAP molecule with the desiredactivity, cytotoxicity, pharmacokinetic properties, and improved tissuedelivery, the SDC-TRAPs are tested for their efficacy in an appropriateanimal model of skin cancer Animal models of skin cancer are well knownin the art. For example, xenograph tumor models using squamous cellcarcinoma, basal cell carcinoma, or melanoma cell lines are used withsubcutaneously implanted tumors. Topical formulations of imiquimod, theeffector molecule, and imiquimod SDC-TRAP molecules are applied. Theactivity of the conjugate is compared to the activity of each imiquimodand the effector molecule alone. Imiquimod SDC-TRAP molecules having oneor more improved properties as compared to either of the parentmolecules are further characterized in other animal systems and humans

The SDC-TRAPs are found to have one or more improved properties in thetreatment of humans including, but not limited to, decreased toxicity,improved dosing schedule, or alternate route of administration.

Example 15 Determining the Permeability of SDC-TRAP Molecules

In order to test the ability SDC-TRAP molecules of the invention toenter cells, an artificial membrane permeability assay (“PAMPA”) can beused. PAMPAs are useful tool for predicting in vivo drug permeabilityfor drugs that enter cells by passive transport mechanisms. LC/MS isused in conjunction with PAMPA assays to determine the ability of theSDC-TRAP molecules of the invention to permeate cells.

Pre-coated PAMPA plates are warmed to room temperature for at least 30minutes prior to adding assay components.

Stock solutions are prepared with the SDC-TRAP molecules to be tested.In order to make a working solution, either 50 μL of 100 μM Stock inDMSO+950 μL of PBS or 50 μL of 200 μM stock is added to 96 deep wellplate, resulting in a 5 μM final concentration or a 10 μM finalconcentration, respectively. 300 μL of the working solution containingeach compound to be tested is added to the appropriate well of a donorPAMPA plate. 200 μL of PBS is added into the corresponding wells of anacceptor PAMPA plates.

The acceptor plate is lowered onto the donor plate and allowed toincubate for five hours. After five hours, a 50 μL aliquot is removedfrom each well of each plate and added into a new 96 deep-well plate.

100 μL of methanol containing an internal standard is added to eachaliquot and analyzed by LC/MS.

In order to calculate the permeability for each SDC-TRAP molecule andthe control molecules, the following formula was used:

Permeability (in unit of cm/s):

$P_{e} = \frac{- {\ln \left\lbrack {1 - {{C_{A}(t)}/C_{equilibrium}}} \right\rbrack}}{A*\left( {{1/V_{D}} + {1/V_{A}}} \right)*t}$$C_{equilibrium} = \frac{{{C_{D}(t)}*V_{D}} + {{C_{A}(t)}*V_{A}}}{V_{D} + V_{A}}$

Mass Retention:

$R = \frac{1 - \left\lbrack {{{C_{D}(t)}*V_{D}} + {{C_{A}(t)}*V_{A}}} \right\rbrack}{C_{0}*V_{D}}$

C₀=initial compound concentration in donor well (mM)C_(D)=compound concentration in donor well at time t. (mM)C_(A)=compound concentration in acceptor well at time t. (mM)V_(D)=donor well volume=0.3 mLV_(A)=acceptor well volume=0.2 mLA=filter area=0.3 cm²t=incubation time=18000 s (5 h)

Example 16 Pharmacodynamics of SDC-TRAP in Xenograft Tumors

SDC-TRAPs can be identified for potent and durable antitumor activity inparticular tumor types suggesting that the drug is slowly cleaved overits residence time in the tumor to provide long term activity. Todetermine whether these effects are through the binding moiety, e.g., anHSP90 binding moiety that results in Hsp90 inhibition, effector agentactivity, i.e., anticancer activity, e.g., kinase inhibitor activity, orboth, the stability of Hsp90 client proteins as well as thephosphorylation of the kinase substrate as a readout for effectormolecule activity. Readouts can be assessed in various tissues as wellas tumor tissue to determine changes in tissue distribution andclearance as well as activity (e.g., to understand mechanism of reducedtoxicity due to better tumor targeting).

Kinetics of Hsp90 inhibition and kinase activity inhibition are assayedat time points relative to the known metabolism of the component agentsof the SDC-TRAP. Selection of time points and concentrations of thevarious agents is well within the ability of those of skill in the art.The analysis demonstrates the relative activity of each of thecomponents of the SDC-TRAP and changes in kinetics and/or tissuedistribution as a result of the conjugate formation.

Example 17 SDC-TRAP-309 Structure and Synthesis

The structure and synthesis of an exemplary SDC-TRAP, SDC-TRAP-309(4-(4-(((S)-1-((1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)amino)-1-oxopropan-2-yl)carbamoyl)phenyl)-5-(2,4-dihydroxy-5-isopropylphenyl)-N-ethyl-4H-1,2,4-triazole-3-carboxamide)is shown below.

To a mixture of4-amino-1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidin-2(1H)-one(527 mg, 2 mmol) and (S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanoicacid (434 mg, 2 mmol) in DMF (5 mL) was added Et₃N (0.92 mL, 6.6 mmol)followed by PyBOP (1.24 g, 2.4 mmol). The reaction mixture was stirredat room temperature overnight then concentrated. The crude residue wastreated with aq. NaHCO₃ then extracted with ethyl acetate. The organiclayer was separated, washed with brine and dried over Na₂SO₄. Themixture was filtered, concentrated and the residue purified twice byISCO using DCM/MeOH (0-10%) as eluent to afford 260 mg (28%) of product.C₁₉H₂₈F₂N₄O₇: 462.19; found: 463.3 (M+H⁺).

The above product (260 mg) was dissolved in DCM (3 mL) and treated withTFA (3 mL). The reaction mixture was stirred at room temperature for 1h, concentrated and dried on high vacuum to get product as a gummy oil.

Example 18 SDC-TRAP-310 Structure and Synthesis

The structure and synthesis of an exemplary SDC-TRAP, SDC-TRAP-310,(4-(4-(4-(((S)-1-((1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamoyl)phenoxy)phenyl)-5-(2,4-dihydroxy-5-isopropylphenyl)-N-ethyl-4H-1,2,4-triazole-3-carboxamide)is shown below.

Step 1 and Step 2(S)-2-amino-N-(1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-3-methylbutanamideTFA salt

To a mixture of4-amino-1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidin-2(1H)-one(527 mg, 2 mmol) and (S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanoicacid (434 mg, 2 mmol) in DMF (5 mL) was added Et₃N (0.92 mL, 6.6 mmol)followed by PyBOP (1.24 g, 2.4 mmol). The reaction mixture was stirredat room temperature overnight then concentrated. The crude residue wastreated with aq. NaHCO₃ then extracted with ethyl acetate. The organiclayer was separated, washed with brine and dried over Na₂SO₄. Themixture was filtered, concentrated and the residue purified twice byISCO using DCM/MeOH (0-10%) as eluent to afford 260 mg (28%) of product.C₁₉H₂₈F₂N₄O₇: 462.19; found: 463.3 (M+H⁺).

The above product (260 mg) was dissolved in DCM (3 mL) and treated withTFA (3 mL). The reaction mixture was stirred at room temperature for 1h, concentrated and dried on high vacuum to get product as a gummy oil.

Step 3

To a mixture of(S)-2-amino-N-(1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-3-methylbutanamideTFA salt (96 mg, 0.2 mmol) and4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-(ethylcarbamoyl)-4H-1,2,4-triazol-4-yl)phenoxy)benzoicacid (120 mg, 0.24 mmol) in DMF (3 mL) was added HATU (92 mg, 0.24 mmol)and DIPEA (0.14 mL, 0.80 mmol).

The reaction mixture was stirred at room temperature overnight thenconcentrated. The crude residue was purified by ISCO (twice) usingDCM/MeOH (0-25%) as eluent to afford 40 mg (23.6%) of the titlecompound. ESMS calculated for C₄₁H₄₄F₂N₈O₁₀: 846.31. found: 847.3(M+H⁺). ¹H NMR (400 MHz, DMSO-d6) δ 11.19 (s, 1H), 10.43 (s, 1H), 9.79(s, 1H), 8.99 (t, J=5.9 Hz, 1H), 8.49 (d, J=7.6 Hz, 1H), 8.28 (d, J=7.6Hz, 1H), 8.03-7.89 (m, 2H), 7.50-7.35 (m, 2H), 7.30 (d, J=7.6 Hz, 1H),7.23-6.95 (m, 4H), 6.69 (s, 1H), 6.35-6.33 (m, 2H), 6.18 (t, J=7.4 Hz,1H), 5.32 (t, J=5.4 Hz, 1H), 4.47 (t, J=7.8 Hz, 1H), 4.31-4.07 (m, 1H),3.98-3.74 (m, 2H), 3.68-3.62 (m, 1H), 3.27-3.12 (m, 2H), 2.98 (p, J=6.9Hz, 1H), 2.20 (h, J=6.7 Hz, 1H), 1.06 (t, J=7.2 Hz, 3H), 0.99 (dd,J=9.5, 6.7 Hz, 6H), 0.93 (d, J=6.9 Hz, 6H).

Example 19 SDC-TRAP-311 Structure and Synthesis

The structure and synthesis of an exemplary SDC-TRAP, SDC-TRAP-311(N—((S)-1-((1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)amino)-3-methyl-1-oxobutan-2-yl)-5-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-(ethylcarbamoyl)-4H-1,2,4-triazol-4-yl)phenoxy)piperidin-1-yl)pyrazine-2-carboxamide)is shown below.

To a mixture of(S)-2-amino-N-(1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-3-methylbutanamideTFA salt (150 mg, 0.31 mmol) and5-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-(ethylcarbamoyl)-4H-1,2,4-triazol-4-yl)phenoxy)piperidin-1-yl)pyrazine-2-carboxylicacid (117 mg, 0.20 mmol) in DMF (3 mL) was added HATU (92 mg, 0.24 mmol)and DIPEA (0.14 mL, 0.80 mmol). The reaction mixture was stirred at roomtemperature overnight then concentrated. The crude residue was purifiedby ISCO using DCM/MeOH (0-25%) as eluent to afford 106 mg (56%) of thetitle compound. ESMS calculated for C₄₄H₅₁F₂N₁₁O₁₀: 931.94. found: 933.0(M+H⁺). ¹H NMR (400 MHz, DMSO-d6) δ 11.30 (s, 1H), 10.69 (s, 1H), 9.75(s, 1H), 8.91 (s, 1H), 8.63 (s, 1H), 8.41 (s, 1H), 8.26 (dd, J=15.0, 8.3Hz, 2H), 7.39-7.20 (m, 3H), 7.17-6.99 (m, 2H), 6.60 (s, 1H), 6.36 (s,1H), 6.33-6.27 (m, 1H), 6.18 (t, J=7.4 Hz, 1H), 5.30-5.26 (m, 1H),4.78-4.74 (m, 1H), 4.61 (dd, J=8.9, 6.6 Hz, 1H), 4.27-4.03 (m, 3H),3.96-3.76 (m, 2H), 3.72-3.56 (m, 1H), 3.25-3.13 (m, 2H), 2.94 (p, J=6.9Hz, 1H), 2.23 (h, J=6.7 Hz, 1H), 2.06 (broad s, 2H), 1.76-1.70 (m, 4H),1.05 (t, J=7.2 Hz, 3H), 0.93 (dd, J=20.2, 6.7 Hz, 6H), 0.85 (d, J=6.9Hz, 6H).

Example 20 SDC-TRAP-312 Structure and Synthesis

The structure and synthesis of an exemplary SDC-TRAP, SDC-TRAP-312(N-(1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-1-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-(ethylcarbamoyl)-4H-1,2,4-triazol-4-yl)phenoxy)benzoyl)piperidine-4-carboxamide)is shown below.

Step 1 and Step 2

To a mixture of4-amino-1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidin-2(1H)-one(527 mg, 2 mmol) and 1-(tert-butoxycarbonyl)piperidine-4-carboxylic acid(458 mg, 2 mmol) in DMF (5 mL) was added HOBt (0.27 g, 2 mmol), Et₃N(0.84 mL, 6 mmol) and PyBOP (1.04 g, 2 mmol). The reaction mixture wasstirred at room temperature overnight then concentrated. The crudeproduct was purified by ISCO using DCM/MeOH (0-10%) as eluent to afford116 mg (12%) of product. C₂₀H₂₈F₂N₄O₇: 474.19; found: 475.3 (M+H⁺).

The above product (116 mg) was dissolved in DCM (3 mL) and treated withTFA (3 mL). The reaction mixture was stirred at room temperature for 1h, concentrated and dried on high vacuum to get title compound as agummy oil.

Step 3

To a mixture ofN-(1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)piperidine-4-carboxamideTFA salt (113 mg, 0.24 mmol) and4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-(ethylcarbamoyl)-4H-1,2,4-triazol-4-yl)phenoxy)benzoicacid (120 mg, 0.24 mmol) in DMF (3 mL) was added HATU (109 mg, 0.28mmol) and DIPEA (0.165 mL, 0.96 mmol). The reaction mixture was stirredat room temperature for 5 h then concentrated. The crude product waspurified by ISCO using DCM/MeOH (0-25%) as eluent to afford 66 mg (32%)of the title compound. ESMS calculated for C₄₂H₄₄F₂N₈O₁₀: 858.84. found:860.0 (M+H⁺). ¹H NMR (400 MHz, DMSO-d6) δ11.07 (s, 1H), 10.41 (s, 1H),9.74 (s, 1H), 8.95 (t, J=5.7 Hz, 1H), 8.25 (d, J=7.6 Hz, 1H), 7.51-7.44(m, 2H), 7.41-7.35 (m, 2H), 7.29 (d, J=7.6 Hz, 1H), 7.14-7.05 (m, 4H),6.68 (s, 1H), 6.35 (s, 1H), 6.30 (d, J=6.5 Hz, 1H), 6.17 (t, J=7.5 Hz,1H), 5.30-5.27 (m, 1H), 4.26-4.10 (m, 1H), 3.89 (dt, J=8.5, 3.0 Hz, 1H),3.86-3.75 (m, 1H), 3.68-3.63 (m, 1H), 3.27-3.10 (m, 3H), 2.97 (h, J=6.8Hz, 3H), 2.81-2.76 (m, 1H), 1.85 (broad s, 2H), 1.59-1.50 (m, 2H), 1.08(dt, J=11.7, 7.1 Hz, 4H), 0.92 (d, J=6.9 Hz, 6H).

Example 21 SDC-TRAP-313 Structure and Synthesis

The structure and synthesis of an exemplary SDC-TRAP, SDC-TRAP-313(N-(1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-4-((4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)benzyl)piperazin-1-yl)methyl)benzamide)is shown below.

Step 14-amino-1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)pyrimidin-2(1H)-one

The compound was prepared following a procedure in Biomacromolecules,2013, 2837-2847, incorporated by reference in its entirety herein. To asolution of4-amino-1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidin-2(1H)-one(1 g, 3.8 mmol) in DMF (23 mL) was added Imidazole (0.78 g, 11.5 mmol)and TBDMSCl (1.5 g, 9.6 mmol). The reaction mixture was stirred at roomtemperature for 1.5 d. The solvent was removed and the residuepartitioned between ethyl acetate and aq. NaHCO₃. The organic layer wasseparated, washed with brine, dried over Na₂SO₄, filtered andconcentrated. The crude product was purified by ISCO using DCM/MeOH(0-10%) as eluent to afford 1.3 g (69%) of the title compound. ESMScalculated for C₂₁H₃₉F₂N₃O₄Si₂: 491.24. found: 492.4 (M+H⁺).

Step 2N-(1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-4-(chloromethyl)benzamide

To a solution of4-amino-1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)pyrimidin-2(1H)-one(100 mg, 0.2 mmol) in DCM (1 mL) at 0° C. was added pyridine (0.48 mmol,40 μL) followed by 4-(chloromethyl)benzoyl chloride (50 mg, 0.24 mmol)in DCM (1 mL). After stirring 45 min, ice-bath removed and furtherstirred at room temperature for 2 h. The solvent was removed and thecrude product purified by ISCO using DCM/MeOH (0-10%) as eluent toafford 120 mg (93%) of product. ESMS calculated for C₂₉H₄₄ClF₂N₃O₅Si₂:643.25. found: 644.6 (M+H⁺).

Step 3N-(1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-4-((4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)benzyl)piperazin-1-yl)methyl)benzamide

To a mixture ofN-(1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-4-(chloromethyl)benzamide(65 mg, 0.1 mmol) and4-(5-hydroxy-4-(4-(piperazin-1-ylmethyl)phenyl)-4H-1,2,4-triazol-3-yl)-6-isopropylbenzene-1,3-diol hydrochloride (75 mg, 0.16 mmol) in DMF (2 mL) was addedDIPEA (0.1 mL, 0.6 mmol) and KI (5 mg). The mixture was heated at 80° C.for 4 h. The solvent was removed and the crude product purified by ISCOusing DCM/MeOH (0-25%) as eluent to afford 91 mg (98%) of the titlecompound. ESMS calculated for C₅₁H₇₀F₂N₈O₈Si₂: 1017.32. found: 1018.4(M+H⁺).

Step 4N-(1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-4-((4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)benzyl)piperazin-1-yl)methyl)benzamide

To a solution ofN-(1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-4-((4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)benzyl)piperazin-1-yl)methyl)benzamide(91 mg, 0.089 mmol) in dry THF (2 mL) was added TBAF (1M, 0.67 mL, 0.67mmol). The mixture was stirred at room temperature for 1.5 h thenquenched with few drops of aq. NH₄Cl. The solvent was removed and thecrude product purified by ISCO using DCM/MeOH (0-25%) as eluent toafford 61 mg (87%) of the title compound. ESMS calculated forC₃₉H₄₂F₂N₈O₈: 788.31. found: 790.0 (M+H⁺).

Example 22 SDC-TRAP-314 Structure and Synthesis

The structure and synthesis of an exemplary SDC-TRAP, SDC-TRAP-314(N-(1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-4-((4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-2-fluorobenzyl)piperazin-1-yl)methyl)benzamideis shown below.

To a mixture ofN-(1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-4-(chloromethyl)benzamide(54 mg, 0.083 mmol) and4-(4-(3-fluoro-4-(piperazin-1-ylmethyl)phenyl)-5-hydroxy-4H-1,2,4-triazol-3-yl)-6-isopropylbenzene-1,3-diolhydrochloride (47 mg, 0.1 mmol) in DMF (2 mL) was added DIPEA (0.14 mL,0.8 mmol) and KI (5 mg). The mixture was heated at 80° C. for 6 h. Thesolvent was removed and the crude product purified by ISCO usingDCM/MeOH (0-25%) as eluent to afford 49 mg (57.6%) of product. ESMScalculated for C₅₁H₆₉F₃N₈O₈Si₂: 1035.31. found: 1036.3 (M+H⁺).

To a solution ofN-(1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-4-((4(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-2-fluorobenzyl)piperazin-1-yl)methyl)benzamide(49 mg, 0.047 mmol) in dry THF (2 mL) was added TBAF (1M, 0.36 mL, 0.36mmol). The mixture was stirred at room temperature for 1.5 h thenquenched with few drops of aq. NH₄Cl. The solvent was removed and thecrude product purified by ISCO using DCM/MeOH (0-25%) as eluent toafford 34 mg (90%) of the title compound. ESMS calculated forC₃₉H₄₁F₃N₈O₈: 806.30. found: 807.6 (M+H⁺). ¹H NMR (400 MHz, DMSO-d6) δ11.97 (s, 1H), 11.32 (s, 1H), 9.63 (s, 1H), 9.39 (s, 1H), 8.31 (d, J=7.7Hz, 1H), 8.06-7.81 (m, 2H), 7.50-7.28 (m, 4H), 7.05 (dd, J=10.9, 2.0 Hz,1H), 6.96 (dd, J=8.2, 2.0 Hz, 1H), 6.85 (s, 1H), 6.34 (d, J=6.5 Hz, 1H),6.27 (s, 1H), 6.20 (t, J=7.4 Hz, 1H), 5.34 (t, J=5.4 Hz, 1H), 4.23 (dd,J=13.6, 6.0 Hz, 1H), 3.91 (dt, J=8.6, 3.1 Hz, 1H), 3.82 (d, J=13.0 Hz,1H), 3.67 (ddd, J=12.7, 5.9, 3.5 Hz, 1H), 3.51 (d, J=16.1 Hz, 4H), 3.00(p, J=6.9 Hz, 1H), 2.36 (broad s, 8H), 0.99 (d, J=6.9 Hz, 6H).

Example 23 SDC-TRAP-315 Structure and Synthesis

The structure and synthesis of an exemplary SDC-TRAP, SDC-TRAP-315(N-(1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)benzyl)piperazine-1-carboxamide is shown below.

Step 1 and Step 2N-(1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)benzyl)piperazine-1-carboxamide

To a solution of4-amino-1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)pyrimidin-2(1H)-one(100 mg, 0.2 mmol) in dry DCM (2 mL) was added pyridine (20 μL, 0.24mmol) and 4-nitrophenyl carbonochloridate (48 mg, 0.24 mmol). Themixture was stirred at 0° C. for 1 h then at room temperature for 5 h.The solvent was removed and the crude product, 4-nitrophenyl(1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamatedried on high vacuum.

To the above product (130 mg, 0.2 mmol) in DMF (3 mL) was added4-(5-hydroxy-4-(4-(piperazin-1-ylmethyl)phenyl)-4H-1,2,4-triazol-3-yl)-6-isopropylbenzene-1,3-diol hydrochloride (90 mg, 0.2 mmol) and DIPEA (0.14 mL, 0.8mmol). The mixture was stirred at room temperature overnight thensolvent removed. The crude product was purified by ISCO using DCM/MeOH(0-25%) as eluent to afford 83 mg (44.8%) of product. ESMS calculatedfor C₄₄H₆₄F₂N₈O₈Si₂: 926.44. found: 927.4 (M+H⁺).

Step 3

To a solution ofN-(1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)benzyl)piperazine-1-carboxamide(83 mg, 0.089 mmol) in dry THF (4 mL) was added TBAF (1M, 0.67 mL, 0.67mmol). The mixture was stirred at room temperature for 1.5 h thenquenched with few drops of aq. NH₄Cl. The solvent was removed and thecrude product purified by ISCO using DCM/MeOH (0-25%) as eluent toafford 55 mg (90%) of the title compound. ESMS calculated forC₃₉H₄₁F₃N₈O₈: 698.26. found: 699.3 (M+H⁺). ¹H NMR (400 MHz, DMSO-d6) δ11.93 (s, 1H), 10.22 (broad s, 1H), 9.63 (s, 1H), 9.42 (s, 1H), 8.10(broad s, 1H), 7.40-7.25 (m, 2H), 7.21-7.07 (m, 2H), 6.92 (broad s, 1H),6.76 (s, 1H), 6.31 (d, J=11.0 Hz, 1H), 6.29 (s, 1H), 6.12 (s, 1H), 5.28(t, J=5.5 Hz, 1H), 4.26-4.18 (m, 1H), 3.92-3.72 (m, 2H), 3.70-3.57 (m,1H), 3.47 (s, 6H), 2.97 (p, J=6.9 Hz, 1H), 2.34 (broad s, 4H), 0.99 (d,J=6.9 Hz, 6H).

Example 24 SDC-TRAP-316 Structure and Synthesis

The structure and synthesis of an exemplary SDC-TRAP, SDC-TRAP-316(4-(4-(((S)-1-((1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamoyl)phenyl)-5-(2,4-dihydroxy-5-isopropylphenyl)-N-ethyl-4H-1,2,4-triazole-3-carboxamide)is shown below.

To a mixture of(S)-2-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-(ethylcarbamoyl)-4H-1,2,4-triazol-4-yl)benzamido)-3-methylbutanoicacid (51 mg, 0.1 mmol) and4-amino-1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidin-2(1H)-one(27 mg, 0.1 mmol) in DMF (2 mL) was added EDCI (25 mg, 0.13 mmol), HOBt(14 mg, 0.1 mmol) and NMM (11 μL, 0.1 mmol). The reaction mixture wasstirred at 55° C. for 24 h. The solvent was removed and the cruderesidue was purified by ISCO (twice) using DCM/MeOH (0-25%) as eluent toafford 8 mg (10%) of the title compound. ESMS calculated forC₃₅H₄₀F₂N₈O₉: 754.29. found: 755.6 (M+H⁺).

Example 25 SDC-TRAP-317 Structure and Synthesis

The structure and synthesis of an exemplary SDC-TRAP, SDC-TRAP-317(N-(1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-1-(2-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)benzyl)piperazin-1-yl)acetyl)piperidine-4-carboxamide)is shown below.

To a mixture ofN-(1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)piperidine-4-carboxamideTFA salt (95 mg, 0.2 mmol) and2-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)benzyl)piperazin-1-yl)aceticacid (94 mg, 0.2 mmol) in DMF (2 mL) was added HATU (92 mg, 0.24 mmol)and DIPEA (0.14 mL, 0.8 mmol). The reaction mixture was stirred at roomtemperature for 5 h then concentrated. The crude residue was purifiedtwice by ISCO using DCM/MeOH (0-25%) as eluent to afford 32 mg (19%) ofthe title compound. ESMS calculated for C₃₉H₄₇F₂N₉O₉: 823.35. found:825.2 (M+H⁺). ¹H NMR (400 MHz, DMSO-d6) δ 11.93 (s, 1H), 11.08 (s, 1H),9.63 (s, 1H), 9.42 (s, 1H), 8.26 (d, J=7.6 Hz, 1H), 7.35-7.25 (m, 3H),7.12 (d, J=8.4 Hz, 2H), 6.76 (s, 1H), 6.34 (d, J=6.5 Hz, 1H), 6.27 (s,1H), 6.17 (t, J=7.5 Hz, 1H), 5.33 (t, J=5.4 Hz, 1H), 4.34 (d, J=12.8 Hz,1H), 4.24-4.07 (m, 2H), 3.89 (dt, J=8.6, 3.1 Hz, 1H), 3.85-3.74 (m, 1H),3.68-3.63 (m, 1H), 3.42 (s, 2H), 3.31-3.19 (m, 2H), 3.01-2.89 (m, 3H),2.77-2.71 (m, 1H), 2.38 (broad s, 8H), 1.81 (broad s, 2H), 1.60-1.49 (m,1H), 1.40-1.28 (m, 1H), 0.94 (d, J=6.9 Hz, 6H).

Example 26 SDC-TRAP-318 Structure and Synthesis

The structure and synthesis of an exemplary SDC-TRAP, SDC-TRAP-318(N-(1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-4-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-2-fluorobenzyl)piperazin-1-yl)-4-oxobutanamide)is shown below.

Step 1

To a solution of4-amino-1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)pyrimidin-2(1H)-one(49 mg, 0.1 mmol) and dihydrofuran-2,5-dione (73 mg, 0.73 mmol, 7.3 eq.)in dry DMF (1 mL) was added DIPEA (0.158 mL, 0.91 mmol, 9.14 eq.). Themixture was stirred at room temperature for 20 h. The solvent wasremoved and the residue purified by ISCO using DCM/MeOH (0-25%) aseluent to afford 60 mg (99%) of product. ESMS calculated forC₂₅H₄₃F₂N₃O₇Si₂: 591.26. found: 592.6 (M+H⁺).

Step 2

To a mixture of4-((1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)amino)-4-oxobutanoicacid (60 mg, 0.1 mmol) and4-(4-(3-fluoro-4-(piperazin-1-ylmethyl)phenyl)-5-hydroxy-4H-1,2,4-triazol-3-yl)-6-isopropylbenzene-1,3-diolhydrochloride (47 mg, 0.1 mmol) in DMF (1 mL) was added EDCI (29 mg,0.15 mmol), HOBt (14 mg, 0.1 mmol) and DIPEA (70 μL, 0.4 mmol). Thereaction mixture was stirred at room temperature overnight. The solventwas removed and the crude residue was purified by ISCO using DCM/MeOH(0-25%) as eluent to afford 79 mg (79%) of impure product. ESMScalculated for C₄₇H₆₇F₃N₈O₉Si₂: 1001.25. found: 1002.3 (M+H⁺).

Step 3

To a solution ofN-(1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-4-(4-((4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-2-fluorobenzyl)piperazin-1-yl)-4-oxobutanamide(79 mg, 0.0788 mmol) in dry THF (4 mL) was added TBAF (1M, 0.8 mL, 0.8mmol). The mixture was stirred at room temperature for 1.5 h thenquenched with few drops of aq. NH₄Cl. The solvent was removed and thecrude product purified by ISCO using DCM/MeOH (0-25%) as eluent toafford 27 mg (45%) of the title compound. ESMS calculated forC₃₅H₃₉F₃N₈O₉: 772.28. found: 773.8 (M+H⁺). ¹H NMR (400 MHz, DMSO-d6) δ11.99 (s, 1H), 11.04 (s, 1H), 9.65 (s, 1H), 9.40 (s, 1H), 8.23 (d, J=7.6Hz, 1H), 7.40 (t, J=8.2 Hz, 1H), 7.25 (d, J=7.6 Hz, 1H), 7.06 (dd,J=10.9, 2.0 Hz, 1H), 6.97 (dd, J=8.1, 2.0 Hz, 1H), 6.87 (s, 1H), 6.33(d, J=6.5 Hz, 1H), 6.27 (s, 1H), 6.17 (t, J=7.5 Hz, 1H), 5.31 (t, J=5.5Hz, 1H), 4.23-4.13 (m, 1H), 3.88 (dt, J=8.6, 3.0 Hz, 1H), 3.85-3.76 (m,1H), 3.68-3.62 (m, 1H), 3.52 (s, 2H), 3.45-3.39 (m, 4H), 3.00 (p, J=6.8Hz, 1H), 2.62-2.59 (m, 4H), 2.39 (broad s, 2H), 2.31 (broad s, 2H), 1.00(d, J=6.9 Hz, 6H).

Example 27 SDC-TRAP-319 Structure and Synthesis

The structure and synthesis of an exemplary SDC-TRAP, SDC-TRAP-319(N-(1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-2-((2-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)benzyl)piperazin-1-yl)-2-oxoethyl)(methyl)amino)acetamide)is shown below.

Step 1

To a solution of4-amino-1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)pyrimidin-2(1H)-one(110 mg, 0.22 mmol) and 4-methylmorpholine-2,6-dione (0.2 g, 1.62 mmol)in dry DMF (1 mL) was added DIPEA (0.35 mL, 2 mmol). The mixture wasstirred at room temperature for 20 h. The solvent was removed and theresidue purified by ISCO using DCM/MeOH (0-25%) as eluent to afford 113mg (81.8%) of product. ESMS calculated for C₂₆H₄₆F₂N₄O₇Si₂: 620.83.found: 621.7 (M+H⁺).

Step 2

To a mixture of2-((2-((1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)amino)-2-oxoethyl)(methyl)amino)aceticacid (56 mg, 0.091 mmol) and4-(5-hydroxy-4-(4-(piperazin-1-ylmethyl)phenyl)-4H-1,2,4-triazol-3-yl)-6-isopropylbenzene-1,3-diolhydrochloride (45 mg, 0.1 mmol) in DMF (1 mL) was added HATU (42 mg,0.1092 mmol) and DIPEA (70 μL, 0.4 mmol). The reaction mixture wasstirred at room temperature for 6 h. The solvent was removed and thecrude residue was purified by ISCO using DCM/MeOH (0-25%) as eluent toafford 40 mg (43.4%) of product. ESMS calculated for C₄₈H₇₁F₂N₉O₉Si₂:1012.30. found: 1013.3 (M+H⁺).

Step 3

To a solution ofN-(1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-2-((2-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)benzyl)piperazin-1-yl)-2-oxoethyl)(methyl)amino)acetamide(40 mg, 0.39 mmol) in dry THF (2 mL) was added TBAF (1M, 0.8 mL, 0.8mmol). The mixture was stirred at room temperature for 1.5 h. Thesolvent was removed and the crude product purified by ISCO usingDCM/MeOH (0-25%) as eluent to afford 25 mg (83%) of the title compound.ESMS calculated for C₃₆H₄₃F₂N₉O₉: 773.32. found: 774.8 (M+H⁺). ¹H NMR(400 MHz, DMSO-d6) δ 11.93, (s, 1H), 10.74 (s, 1H), 9.60 (s, 1H), 9.41(s, 1H), 8.28 (d, J=7.5 Hz, 1H), 7.37-7.25 (m, 3H), 7.14 (d, J=8.4 Hz,2H), 6.78 (s, 1H), 6.33 (d, J=6.5 Hz, 1H), 6.26 (s, 1H), 6.18 (t, J=7.4Hz, 1H), 5.32 (t, J=5.4 Hz, 1H), 4.24-4.14 (m, 1H), 3.96-3.76 (m, 2H),3.74-3.59 (m, 1H), 3.46 (broad s, 8H), 3.31 (s, 2H), 2.97 (p, J=6.9 Hz,1H), 2.37 (s, 4H), 2.30 (s, 3H), 0.95 (d, J=6.9 Hz, 6H).

Example 28 SDC-TRAP-320 Structure and Synthesis

The structure and synthesis of an exemplary SDC-TRAP, SDC-TRAP-320(N-(1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-2-((2-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-2-fluorobenzyl)piperazin-1-yl)-2-oxoethyl)(methyl)amino)acetamide)is shown below.

Step 1 and Step 2

To a mixture of2-((2-((1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)amino)-2-oxoethyl)(methyl)amino)acetic acid (56 mg, 0.091mmol) and4-(4-(3-fluoro-4-(piperazin-1-ylmethyl)phenyl)-5-hydroxy-4H-1,2,4-triazol-3-yl)-6-isopropylbenzene-1,3-diol hydrochloride (47 mg, 0.1 mmol) in DMF (1 mL) was addedHATU (42 mg, 0.1092 mmol) and DIPEA (70 μL, 0.4 mmol). The reactionmixture was stirred at room temperature for 6 h. The solvent was removedand the crude residue was purified by ISCO using DCM/MeOH (0-25%) aseluent to afford 58 mg (62.3%) of product. ESMS calculated forC₄₈H₇₀F₃N₉O₉Si₂: 1030.29. found: 1031.4 (M+H⁺).

Step 3

To a solution ofN-(1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-2-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-2-fluorobenzyl)piperazin-1-yl)-2-oxoethyl)(methyl)amino)acetamide(58 mg, 0.056 mmol) in dry THF (2 mL) was added TBAF (1M, 0.8 mL, 0.8mmol). The mixture was stirred at room temperature for 1.5 h. Thesolvent was removed and the crude product purified by ISCO usingDCM/MeOH (0-25%) as eluent to afford 39 mg (88%) of the title compound.ESMS calculated for C₃₆H₄₂F₃N₉O₉: 801.31. found: 802.4 (M+H⁺). ¹H NMR(400 MHz, DMSO-d6) δ 11.98 (s, 1H), 10.74 (s, 1H), 9.63 (s, 1H), 9.39(s, 1H), 8.28 (d, J=7.6 Hz, 1H), 7.40 (t, J=8.2 Hz, 1H), 7.28 (d, J=7.5Hz, 1H), 7.06 (dd, J=10.8, 2.0 Hz, 1H), 6.97 (dd, J=8.1, 2.0 Hz, 1H),6.87 (s, 1H), 6.33 (d, J=6.5 Hz, 1H), 6.27 (s, 1H), 6.18 (t, J=7.4 Hz,1H), 5.32 (t, J=5.4 Hz, 1H), 4.28-4.10 (m, 1H), 3.95-3.76 (m, 2H),3.68-3.63 (m, 1H), 3.52 (s, 2H), 3.45-3.39 (m, 6H), 3.31 (s, 2H), 3.00(p, J=6.9 Hz, 1H), 2.44-2.25 (m, 7H), 1.00 (d, J=6.9 Hz, 6H).

Example 29 SDC-TRAP-321 Structure and Synthesis

The structure and synthesis of an exemplary SDC-TRAP, SDC-TRAP-321(N-(1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-2-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)benzyl)piperazin-1-yl)acetamide)is shown below.

Step 1

To a solution of4-amino-1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)pyrimidin-2(1H)-one(245 mg, 0.5 mmol) in DCM (3 mL) at 0° C. was added pyridine (0.1 mL,1.2 mmol) followed by 2-chloroacetyl chloride (48 μL, 0.6 mmol) in DCM(1 mL). The reaction mixture was allowed to reach room temperature over5 h. The solvent was removed and the crude product purified by ISCOusing DCM/MeOH (0-10%) as eluent to afford 0.28 g (99%) of product. ESMScalculated for C₂₃H₄₀C₁F₂N₃O₅Si₂: 567.22. found: 568.6 (M+H⁺).

Step 2

To a mixture ofN-(1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-2-chloroacetamide(93 mg, 0.163 mmol) and4-(5-hydroxy-4-(4-(piperazin-1-ylmethyl)phenyl)-4H-1,2,4-triazol-3-yl)-6-isopropylbenzene-1,3-diol hydrochloride (116 mg, 0.26 mmol) in DMF (2 mL) was addedDIPEA (0.16 mL, 0.92 mmol) and KI (5 mg). The mixture was heated at 70°C. for 5 h. The reaction mixture was diluted with the ethyl acetate,washed with water and brine. The organic layer dried over Na₂SO₄,filtered, concentrated and the crude product purified by ISCO usingDCM/MeOH (0-25%) as eluent to afford 89 mg (58%) of product. ESMScalculated for C₄₅H₆₆F₂N₈O₈Si₂: 941.22. found: 942.3 (M+H⁺).

Step 3

To a solution ofN-(1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-2-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)benzyl)piperazin-1-yl)acetamide(89 mg, 0.094 mmol) in dry THF (2 mL) was added TBAF (1M, 0.71 mL, 0.71mmol). The mixture was stirred at room temperature for 1.5 h. Thesolvent was removed and the crude product purified by ISCO usingDCM/MeOH (0-25%) as eluent to afford 60 mg (90%) of the title compound.ESMS calculated for C₃₃H₃₈F₂N₈O₈: 712.28. found: 713.70 (M+H⁺). ¹H NMR(400 MHz, DMSO-d6) δ 11.93 (s, 1H), 10.45 (s, 1H), 9.60 (s, 1H), 9.41(s, 1H), 8.29 (d, J=7.5 Hz, 1H), 7.29 (t, J=7.9 Hz, 3H), 7.13 (d, J=8.4Hz, 2H), 6.77 (s, 1H), 6.33 (d, J=6.5 Hz, 1H), 6.26 (s, 1H), 6.17 (t,J=7.4 Hz, 1H), 5.32 (t, J=5.5 Hz, 1H), 4.24-4.14 (m, 1H), 3.96-3.74 (m,2H), 3.68-3.63 (m, 1H), 3.45 (broad s, 4H), 3.22 (broad s, 4H), 2.96 (p,J=6.8 Hz, 1H), 2.40 (broad s, 4H), 1.00 (d, J=6.9 Hz, 6H).

Example 30 SDC-TRAP-322 Structure and Synthesis

The structure and synthesis of an exemplary SDC-TRAP, SDC-TRAP-322(N-(1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-5-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-2-fluorobenzyl)piperazin-1-yl)-3,3-dimethyl-5-oxopentanamide)is shown below.

Step 1

To a solution of4-amino-1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)pyrimidin-2(1H)-one(110 mg, 0.22 mmol) and 4,4-dimethyldihydro-2H-pyran-2,6(3H)-dione (0.23g, 1.62 mmol) in dry DMF (1 mL) was added DIPEA (0.35 mL, 2 mmol). Themixture was stirred at room temperature for 20 h. The solvent wasremoved and the residue purified by ISCO using DCM/MeOH (0-25%) aseluent to afford 141 mg (99%) of product. ESMS calculated forC₂₈H₄₉F₂N₃O₇Si₂: 633.87. found: 634.7 (M+H⁺).

Step 2

To a mixture of5-((1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)amino)-3,3-dimethyl-5-oxopentanoicacid (141 mg, 0.22 mmol) and4-(4-(3-fluoro-4-(piperazin-1-ylmethyl)phenyl)-5-hydroxy-4H-1,2,4-triazol-3-yl)-6-isopropylbenzene-1,3-diolhydrochloride (104 mg, 0.22 mmol) in DMF (2 mL) was added HATU (103 mg,0.27 mmol) and DIPEA (0.16 mL, 0.9 mmol). The reaction mixture wasstirred at room temperature for 6 h. The solvent was removed and thecrude residue was purified by ISCO using DCM/MeOH (0-25%) as eluent toafford 45 mg (23%) of product. ESMS calculated for C₅₀H₇₃F₃N₈O₉Si₂:1043.3. found: 1044.4 (M+H⁺).

Step 3

To a solution ofN-(1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-5-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-2-fluorobenzyl)piperazin-1-yl)-3,3-dimethyl-5-oxopentanamide(45 mg, 0.043 mmol) in dry THF (3 mL) was added TBAF (1M, 0.7 mL, 0.7mmol). The mixture was stirred at room temperature for 1.5 h. Thesolvent was removed and the crude product purified by ISCO usingDCM/MeOH (0-25%) as eluent to afford 39 mg (90%) of the title compound.ESMS calculated for C₃₈H₄₅F₃N₈O₉: 814.33. found: 815.81 (M+H⁺). ¹H NMR(400 MHz, DMSO-d6) δ11.98 (s, 1H), 11.12 (s, 1H), 9.63 (s, 1H), 9.40 (s,1H), 8.24 (d, J=7.6 Hz, 1H), 7.40 (t, J=8.2 Hz, 1H), 7.30 (d, J=7.6 Hz,1H), 7.07 (dd, J=10.8, 2.0 Hz, 1H), 6.97 (dd, J=8.2, 2.0 Hz, 1H), 6.87(s, 1H), 6.32 (d, J=6.5 Hz, 1H), 6.27 (s, 1H), 6.17 (t, J=7.4 Hz, 1H),5.31 (t, J=5.4 Hz, 1H), 4.24-4.14 (m, 1H), 3.94-3.76 (m, 2H), 3.68-3.62(m, 1H), 3.51 (broad s, 6H) 3.00 (p, J=6.9 Hz, 1H), 2.48 (s, 2H), 2.41(s, 2H), 2.38 (broad s, 2H), 2.32 (broad s, 2H), 1.04 (s, 6H), 1.00 (d,J=6.9 Hz, 6H).

Example 31 SDC-TRAP-323 Structure and Synthesis

The structure and synthesis of an exemplary SDC-TRAP, SDC-TRAP-323(N-(1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-2-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-2-fluorobenzyl)piperazin-1-yl)acetamide)is shown below.

The above compound was prepared following the protocols described forSDC-TRAP-321. C₃₃H₃₇F₃N₈O₈: 730.27. found: 731.70 (M+H⁺). ¹H NMR (400MHz, DMSO-d6) δ 11.98 (s, 1H), 10.45 (s, 1H), 9.62 (s, 1H), 9.40 (s,1H), 8.29 (d, J=7.6 Hz, 1H), 7.37 (t, J=8.2 Hz, 1H), 7.27 (d, J=7.6 Hz,1H), 7.05 (dd, J=10.8, 2.1 Hz, 1H), 6.96 (dd, J=8.1, 2.0 Hz, 1H), 6.87(s, 1H), 6.33 (d, J=6.5 Hz, 1H), 6.27 (s, 1H), 6.17 (t, J=7.4 Hz, 1H),5.32 (s, 1H), 4.24 (m, 1H), 3.94-3.75 (m, 2H), 3.68-3.63 (m, 1H), 3.50(broad s, 4H), 3.32-3.08 (m, 4H), 3.00 (p, J=6.8 Hz, 1H), 2.43 (s, 4H),1.00 (d, J=6.9 Hz, 6H).

Example 32 SDC-TRAP-324 Structure and Synthesis

The structure and synthesis of an exemplary SDC-TRAP, SDC-TRAP-324(4-(4-(((S)-1-((1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamoyl)phenyl)-5-(2,4-dihydroxy-5-isopropylphenyl)-N-ethyl-4H-1,2,4-triazole-3-carboxamide)is shown below.

To a solution of(S)-2-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-(ethylcarbamoyl)-4H-1,2,4-triazol-4-yl)benzamido)-4-methylpentanoicacid (53 mg, 0.2 mmol) in dry DMF (2 mL) was added EDC (29 mg, 0.15mmol) and 1-hydroxypyrrolidine-2,5-dione (18 mg, 0.15 mmol). The mixturewas stirred at room temperature overnight and treated with4-amino-1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidin-2(1H)-one(52 mg, 0.2 mmol). The reaction mixture was stirred at 50° C. 2.5 days.The solvent was removed and the crude residue was purified by ISCO usingDCM/MeOH (0-25%) as eluent to afford 29 mg (37.7%) of the titlecompound. ESMS calculated for C₃₆H₄₂F₂N₈O₉: 768.30. found: 769.6 (M+H⁺).¹H NMR (400 MHz, DMSO-d₆) δ 11.30 (d, J=7.7 Hz, 1H), 9.98 (s, 1H), 9.68(s, 1H), 9.04 (t, J=5.8 Hz, 1H), 8.74 (d, J=7.4 Hz, 1H), 8.27 (d, J=7.6Hz, 1H), 7.97-7.83 (m, 2H), 7.49-7.37 (m, 2H), 7.26 (dd, J=7.6, 3.7 Hz,1H), 6.75 (s, 1H), 6.34 (dd, J=6.5, 1.9 Hz, 1H), 6.28 (s, 1H), 6.18 (t,J=7.3 Hz, 1H), 5.31 (q, J=5.4 Hz, 1H), 4.70-4.66 (m, 1H), 4.25-4.10 (m,1H), 3.95-3.75 (m, 2H), 3.72-3.57 (m, 1H), 3.26-3.11 (m, 2H), 3.04-2.89(m, 1H), 1.87-1.68 (m, 2H), 1.62-1.48 (m, 1H), 1.06 (t, J=7.2 Hz, 3H),0.98-0.81 (m, 12H).

Example 33 SDC-TRAP-325 Structure and Synthesis

The structure and synthesis of an exemplary SDC-TRAP, SDC-TRAP-325(N-(1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-4-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)benzyl)piperazin-1-yl)-4-oxobutanamide)is shown below.

To a mixture of4-((1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)amino)-4-oxobutanoicacid (143 mg, 0.24 mmol) and4-(5-hydroxy-4-(4-(piperazin-1-ylmethyl)phenyl)-4H-1,2,4-triazol-3-yl)-6-isopropylbenzene-1,3-diol hydrochloride (107 mg, 0.24 mmol) in DMF (1 mL) was addedHATU (110 mg, 0.29 mmol) and DIPEA (168 μL, 0.96 mmol). The reactionmixture was stirred at room temperature overnight. The solvent wasremoved and the crude residue was purified by ISCO using DCM/MeOH(0-25%) as eluent to afford 150 mg of impure product. ESMS calculatedfor C₄₇H₆₈F₂N₈O₉Si₂: 983.26. found: 984.4 (M+H+).

To a solution ofN-(1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-4-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)benzyl)piperazin-1-yl)-4-oxobutanamide(150 mg, 0.15 mmol) in dry THF (2 mL) was added TBAF (1M, 1 mL, 1 mmol).The mixture was stirred at room temperature for 1.5 h. The solvent wasremoved and the crude product purified by ISCO using DCM/MeOH (0-25%) aseluent to afford 9 mg of the title compound. ESMS calculated forC₃₅H₄₀F₂N₈O₉: 754.29. found: 755.4 (M+H⁺).

Example 34 SDC-TRAP-326 Structure and Synthesis

The structure and synthesis of an exemplary SDC-TRAP, SDC-TRAP-326(3-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)benzyl)piperazin-1-yl)-3-oxopropyl(1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamate)is shown below.

Step 1

To a solution of4-amino-1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)pyrimidin-2(1H)-one(245 mg, 0.5 mmol) in dry DCM (2.5 mL) at 0° C. was added pyridine (0.16mL, 2 mmol) and phosgene in toluene (1.4 mL, 2 mmol). The mixture wasstirred at 0° C. for 1 h. The solvent was removed and the product driedon high vacuum. To the above product in toluene (5 mL) was addedtert-butyl 3-hydroxypropanoate (0.15 mL, 1 mmol) and heated at 100° C.for 1.5 h. The solvent was removed and the crude residue purified byISCO using DCM/MeOH (0-10%) as eluent to afford 283 mg (85%) of product.ESMS calculated for C₂₉H₅₁F₂N₃O₈Si₂: 663.90. found: 664.7 (M+H+).

Step 2

To a solution of tert-butyl3-(((1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamoyl)oxy)propanoate(0.28 g, 0.421 mmol) in dry DCM (3 mL) was added slowly TFA (3 mL). Themixture was stirred at room temperature for 2 h, solvent was removed andthe product (250 mg, 98%) dried on high vacuum. ESMS calculated forC₂₅H₄₃F₂N₃O₈Si₂: 607.79. found: 608.4 (M+H+).

Step 3& 4

To a mixture of3-(((1((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamoyl)oxy)propanoicacid (95 mg, 0.15 mmol) and4-(5-hydroxy-4-(4-(piperazin-1-ylmethyl)phenyl)-4H-1,2,4-triazol-3-yl)-6-isopropylbenzene-1,3-diolhydrochloride (70 mg, 0.15 mmol) in DMF (2 mL) was added EDCI (45 mg,0.24 mmol), HOBt (21 mg, 0.15 mmol) and DIPEA (0.1 mL, 0.6 mmol). Thereaction mixture was stirred at room temperature overnight. The solventwas removed and the crude residue was purified by ISCO using DCM/MeOH(0-25%) as eluent to get 25 mg of product.

The above product was dissolved in THF (2 mL) and treated with TBAF (1M,0.2 mL, 0.2 mmol). The mixture was stirred at room temperature for 1.5h. The solvent was removed and the crude product purified by ISCO usingDCM/MeOH (0-25%) as eluent to afford of the title compound (21 mg). ESMScalculated for C₃₅H₄₀F₂N₈O₁₀: 770.28. found: 771.4 (M+H⁺). ¹H NMR (400MHz, DMSO-d6) δ 11.93 (s, 1H), 10.86 (s, 1H), 9.61 (s, 1H), 9.40 (s,1H), 8.22 (d, J=7.6 Hz, 1H), 7.31 (d, J=8.4 Hz, 2H), 7.18-7.12 (m, 2H),7.09 (d, J=7.6 Hz, 1H), 6.78 (s, 1H), 6.32 (d, J=6.5 Hz, 1H), 6.26 (s,1H), 6.16 (t, J=7.5 Hz, 1H), 5.31 (t, J=5.5 Hz, 1H), 4.33 (t, J=6.4 Hz,2H), 4.26-4.10 (m, 1H), 3.90-3.86 (m, 1H), 3.81-3.78 (m, 1H), 3.69-3.59(m, 1H), 3.46-3.44 (m, 6H), 2.97 (p, J=6.9 Hz, 1H), 2.71 (t, J=6.4 Hz,2H), 2.37-2.325 (m, 4H), 0.95 (d, J=6.9 Hz, 6H).

Example 35 SDC-TRAP-327 Structure and Synthesis

The structure and synthesis of an exemplary SDC-TRAP, SDC-TRAP-327(3-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-2-fluorobenzyl)piperazin-1-yl)-3-oxopropyl(1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamate)is shown below.

The above compound was prepared following protocols described forSDC-TRAP-326. ESMS calculated for C₃₅H₃₉F₃N₈O₁₀: 788.27. found: 789.6(M+H⁺). ¹H NMR (400 MHz, DMSO-d6) δ 11.99 (s, 1H), 10.86 (s, 1H), 9.66(s, J=2.7 Hz, 1H), 9.41 (s, J=1.5 Hz, 1H), 8.22 (d, J=7.7 Hz, 1H), 7.39(t, J=8.2 Hz, 1H), 7.14-7.03 (m, 2H), 6.97 (dd, J=8.1, 2.0 Hz, 1H), 6.87(s, 1H), 6.33 (d, J=6.5 Hz, 1H), 6.27 (s, 1H), 6.16 (t, J=7.4 Hz, 1H),5.32 (broad s, 1H), 4.32 (t, J=6.4 Hz, 2H), 4.28-4.10 (m, 1H), 3.88 (dt,J=8.6, 3.0 Hz, 1H), 3.82-3.79 (m, 1H), 3.68-3.62 (m, 1H), 3.52 (s, 2H),3.43 (broad s, 4H), 3.00 (h, J=6.8 Hz, 1H), 2.71 (t, J=6.5 Hz, 2H), 2.35(dt, J=23.1, 4.7 Hz, 4H), 1.00 (d, J=6.9 Hz, 6H).

Example 36 SDC-TRAP-328 Structure and Synthesis

The structure and synthesis of an exemplary SDC-TRAP, SDC-TRAP-328(N-(1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-2-((4-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)benzyl)piperazin-1-yl)-1,3,5-triazin-2-yl)amino)acetamide)is shown below.

Step 1

4-amino-1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidin-2(1H)-one(131 mg, 0.5 mmol) and 2,5-dioxopyrrolidin-1-yl2-((tert-butoxycarbonyl)amino)acetate (148 mg, 0.5 mmol) in dry DMF (5mL) heated at 45° C. for 20 h. The solvent was removed and the crudeproduct purified by ISCO using DCM/MeOH (0-10%) as eluent to affordproduct (90 mg, 42.8%). ESMS calculated for C₁₆H₂₂F₂N₄O₇: 420.37. found:421.5 (M+H+).

Step 2

To a solution of tert-butyl(2-((1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)amino)-2-oxoethyl)carbamate(90 mg, 0.214 mmol) in dry DCM (3 mL) was added slowly TFA (1.5 mL). Themixture was stirred at room temperature for 1 h, solvent was removed andthe product (90 mg, 98%) dried on high vacuum.

Step 3 & 4

To a solution of2-amino-N-(1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)acetamide,TFA salt (90 mg, 0.21 mmol) in dry THF (3 mL) was added2,4-dichloro-1,3,5-triazine (32 mg, 0.21 mmol) and DIPEA (93 μL, 0.53mmol) at 0° C. The reaction mixture was stirred at this temperature for2 h and concentrated, dried on high vacuum.

The above crude product2-((4-chloro-1,3,5-triazin-2-yl)amino)-N-(1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)acetamide (0.21 mmol) was dissolved in DMF (3mL) then treated with4-(5-hydroxy-4-(4-(piperazin-1-ylmethyl)phenyl)-4H-1,2,4-triazol-3-yl)-6-isopropylbenzene-1,3-diolhydrochloride (95 mg, 0.21 mmol) and DIPEA (93 μL, 0.53 mmol). Thereaction mixture was stirred at room temperature for 2 h. The solventwas removed and the crude product purified by ISCO using DCM/MeOH(0-10%) as eluent to afford impure product. The impure product waspurified again on reverse phase column chromatography using ACN/H₂O(0.1% HCOOH) as eluent to get 32 mg of product. ESMS calculated forC₃₆H₄₀F₂N₁₂O₈: 806.31. found: 807.8 (M+H+). ¹H NMR (400 MHz, DMSO-d6) δ11.93 (s, 1H), 11.17 (s, 1H), 9.61 (s, 1H), 9.41 (s, 1H), 8.27-8.24 (m,1H), 8.18-8.04 (m, 1H), 7.71 (t, J=6.0 Hz, 1H), 7.39-7.10 (m, 5H), 6.76(d, J=6.9 Hz, 1H), 6.33 (broad s, 1H), 6.27 (s, 1H), 6.18 (t, J=7.4 Hz,1H), 5.30 (broad s, 1H), 4.28-3.98 (m, 3H), 3.89 (dt, J=8.5, 3.0 Hz,1H), 3.81-3.78 (m, 1H), 3.76-3.54 (m, 6H), 2.96 (h, J=6.9 Hz, 1H), 2.38(broad s, 2H), 2.27 (broad s, 3H), 0.93 (d, J=6.9 Hz, 6H).

Example 37 SDC-TRAP-329 Structure and Synthesis

The structure and synthesis of an exemplary SDC-TRAP, SDC-TRAP-329(4-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-(ethylcarbamoyl)-4H-1,2,4-triazol-4-yl)benzoyl)piperazin-1-yl)-2-methoxyphenyl(1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamate)is shown below.

¹H NMR (400 MHz, DMSO-d6) δ11.34 (s, 1H), 10.22 (s, 1H), 9.74 (s, 1H),9.02 (t, J=5.9 Hz, 1H), 8.26 (d, J=7.6 Hz, 1H), 7.54-7.49 (m, 2H),7.47-7.35 (m, 2H), 7.04 (t, J=8.0 Hz, 2H), 6.80-6.67 (m, 2H), 6.55-6.47(m, 1H), 6.33 (d, J=6.9 Hz, 2H), 6.18 (t, J=7.3 Hz, 1H), 5.30 (t, J=5.5Hz, 1H), 4.27-4.06 (m, 2H), 3.78 (s, 9H), 3.18 (dd, J=12.5, 6.0 Hz, 6H),3.04-2.89 (m, 1H), 1.06 (t, J=7.2 Hz, 3H), 0.91 (d, J=6.8 Hz, 6H). ESMScalculated (C₄₂H₄₅F₂N₉O₁₁): 889.3. found: 890.4 (M+H).

Example 38 SDC-TRAP-330 Structure and Synthesis

The structure and synthesis of an exemplary SDC-TRAP, SDC-TRAP-330(4-(4-(4-((3-(2,4-dihydroxy-5-isopropylphenyl)-5-(ethylcarbamoyl)-4H-1,2,4-triazol-4-yl)benzoyl)piperazin-1-yl)-2-methylphenyl(1-((2S,4S,5S)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamate)is shown below.

ESMS calculated (C₄₂H₄₅F₂N₉O₁₀): 873.3. found: 874.4 (M+H).

Example 39 SDC-TRAP-331 Structure and Synthesis

The structure and synthesis of an exemplary SDC-TRAP, SDC-TRAP-331(N-(1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-5-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-2-fluorobenzyl)piperazin-1-yl)pyrazine-2-carboxamide)is shown below.

To the solution of TBS protected Gemcitabine (0.49 g, 1.0 mmol) in DCM(20 mL) was added 5-chloropyrazine-2-carbonyl chloride (0.2 g, 1.1 mmol)and TEA (0.16 mL, 1.1 mmol). The reaction was stirred at roomtemperature for 60 min before it was concentrated. The columnchromatography gave the product (0.51 g, 98%).

To the solution of starting material (0.05 g, 0.096 mmol) in DMF (3 mL)was added amine (0.05 g, 0.1 mmol) and K₂CO₃ (0.041 g, 0.3 mmol). Thereaction was heated in the microwave at 85° C. for 60 min before H₂O (3mL) was added. The mixture was extracted with EtOAc (15 mL) and theorganic phase was dried over Na₂SO₄ and concentrated. The columnchromatography gave the product (0.06 g, 61%).

To the solution of starting material (0.03 g, 0.03 mmol) in THF (2 mL)was added TBAF (1M in THF, 0.09 mL, 0.09 mmol). The reaction was stirredfor 60 min before the reaction was concentrated. The columnchromatography gave SDC-TRAP-331 (0.02 g, 84%).

(N-(1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-5-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-2-fluorobenzyl)piperazin-1-yl)pyrazine-2-carboxamide);¹H NMR (400 MHz, DMSO-d₆) δ 11.98 (s, 1H), 10.07 (s, 1H), 9.62 (s, 1H),9.39 (s, 1H), 8.76 (d, J=1.0 Hz, 1H), 8.44-8.29 (m, 2H), 7.51-7.38 (m,2H), 7.08 (dd, J=10.8, 2.0 Hz, 1H), 6.99 (dd, J=8.2, 2.0 Hz, 1H), 6.87(s, 1H), 6.33 (d, J=6.5 Hz, 1H), 6.27 (s, 1H), 6.20 (t, J=7.4 Hz, 1H),5.33 (t, J=5.4 Hz, 1H), 4.21 (ddd, J=19.6, 12.9, 7.5 Hz, 1H), 3.95-3.63(m, 8H), 3.58 (s, 2H), 3.00 (p, J=6.9 Hz, 1H), 0.99 (d, J=6.9 Hz, 6H);ESMS calculated (C₃₆H₃₇F₃N₁₀O₈): 794.3. found: 795.8 (M+H).

Example 40 SDC-TRAP-332 Structure

The structure of an exemplary SDC-TRAP, SDC-TRAP-332, is shown below.

Example 41 SDC-TRAP-333 Structure

The structure of an exemplary SDC-TRAP, SDC-TRAP-333, is shown below.

Example 42 SDC-TRAP-334 Structure

The structure of an exemplary SDC-TRAP, SDC-TRAP-334, is shown below.

FIG. 3 shows a summary of the structure, physical properties, type oflinkage, Her2 IC₅₀ (nM) and cytotoxicity (BT474 IC50 nM) for theexemplary SDC-TRAPs as described herein.

All publications, patent applications, patents, and other documentscited herein are incorporated by reference in their entirety. In case ofconflict, the present specification, including definitions, willcontrol.

The specification should be understood as disclosing and encompassingall possible permutations and combinations of the described aspects,embodiments, and examples unless the context indicates otherwise. One ofordinary skill in the art will appreciate that the invention can bepracticed by other than the summarized and described aspect,embodiments, and examples, which are presented for purposes ofillustration, and that the invention is limited only by the followingclaims.

1. A binding moiety-drug conjugate (SDC-TRAP) comprising a bindingmoiety and an effector moiety.
 2. The SDC-TRAP of claim 1, wherein thebinding moiety interacts with a protein that is overexpressed incancerous cells compared to normal cells.
 3. The SDC-TRAP of claim 2,wherein the protein is a chaperonin protein.
 4. The SDC-TRAP of claim 3,wherein the chaperonin is Hsp90.
 5. The SDC-TRAP of claim 4, wherein thebinding moiety is an Hsp90 ligand or a prodrug thereof.
 6. The SDC-TRAPof claim 5, wherein the Hsp90 ligand is an Hsp90 inhibitor.
 7. TheSDC-TRAP of claim 6, wherein the Hsp90 inhibitor is selected from thegroup consisting of geldanamycins, macbecins, tripterins, tanespimycins,and radicicols.
 8. The SDC-TRAP of claim 1, wherein the effector moietyis selected from the group consisting of a cytotoxic moiety, atherapeutic moiety and an imaging moiety. 9.-13. (canceled)
 14. TheSDC-TRAP of claim 5, wherein the molecular weight of the SDC-TRAP isless than about 1600 Daltons, less than about 1200 Daltons, less thanabout 800 Daltons, or less than about 400 Daltons. 15.-18. (canceled)19. The SDC-TRAP of claim 1, wherein the binding moiety and the effectormoiety are covalently attached.
 20. The SDC-TRAP of claim 19, whereinthe binding moiety and the effector moiety are covalently attached by alinker. 21.-372. (canceled)
 373. A pharmaceutical composition comprisinga therapeutically effective amount of at least one SDC-TRAP, and atleast one pharmaceutical excipient.
 374. The pharmaceutical compositionof claim 373, wherein the SDC-TRAP comprises the SDC-TRAP of claim 1.375.-416. (canceled)
 417. A kit for imaging, diagnosing, and/orselecting a subject comprising at least one SDC-TRAP and instruction foradministering an effective amount of at least one SDC-TRAP to thesubject, thereby imaging, diagnosing, and/or selecting the subject. 418.(canceled)
 419. A binding moiety-drug conjugate (SDC-TRAP) comprising abinding moiety and an effector moiety substantially as presented in anyone of SDC-TRAP-0001-0420.
 420. A pharmaceutical composition comprisinga therapeutically effective amount of at least one ofSDC-TRAP-0001-0420, and at least one pharmaceutical excipient.
 421. Amethod for treating a subject having a cancer comprising administering atherapeutically effective amount of at least one SDC-TRAP to thesubject, thereby treating the cancer, wherein the SDC-TRAP comprises theSDC-TRAP of claim
 1. 422.-427. (canceled)
 428. A method for treating asubject having chronic bronchitis comprising administering atherapeutically effective amount of at least one SDC-TRAP to thesubject, thereby treating the chronic bronchitis, wherein the SDC-TRAPcomprises the SDC-TRAP of claim
 1. 429. A method for treating a subjecthaving asthma comprising administering a therapeutically effectiveamount of at least one SDC-TRAP to the subject, thereby treating theasthma, wherein the SDC-TRAP comprises the SDC-TRAP of claim
 1. 430. Amethod for treating a subject having actinic keratosis comprisingadministering a therapeutically effective amount of at least oneSDC-TRAP to the subject, thereby treating the actinic keratosis, whereinthe SDC-TRAP comprises the SDC-TRAP of claim 1.